http://www.plantmethods.com The latest research articles published by Plant Methods 2012-05-17T00:00:00Z Background: We have developed a new analytical approach for isolation and quantification of cytokinins (CK) in minute amounts of fresh plant material, which combines a simple one-step purification with ultra-high pressure liquid chromatography-fast scanning tandem mass spectrometry. Results: Plant tissue samples (1-5 mg FW) were purified by stop-and-go-microextraction (StageTip purification), which previously has only been applied for clean-up and pre-concentration of peptides. We found that a combination of two reverse phases and one cation-exchange phase, was the best tool, giving a total extraction recovery higher than 80%. The process was completed by a single chromatographic analysis of a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) in 24.5 minutes using an analytical column packed with sub-2-microne particles. In multiple reaction monitoring mode, the detection limits ranged from 0.05 to 5 fmol and the linear ranges for most cytokinins were at least five orders of magnitude. The StageTip purification was validated and optimized using samples of Arabidopsis thaliana seedlings, roots and shoots where eighteen cytokinins were successfully determined. Conclusions: The combination of microextraction with one-step high-throughput purification provides fast, effective and cheap sample preparation prior to qualitative and quantitative measurements. Our procedure can be used after modification also for other phytohormones, depending on selectivity, affinity and capacity of the selected sorbents. http://www.plantmethods.com/content/8/1/17 Jana Svacinova Ondrej Novak Lenka Plackova Rene Lenobel Josef Holik Miroslav Strnad Karel Dolezal Plant Methods 2012, null:17 2012-05-17T00:00:00Z doi:10.1186/1746-4811-8-17 A microscale high-throughput method for the analysis of the cytokinin content of plant tissues. /content/figures/1746-4811-8-17-toc.gif Plant Methods 1746-4811 ${item.volume} 17 2012-05-17T00:00:00Z PDF Background: Seed transmission constitutes a major component of the parasitic cycle for several fungal pathogens. However, very little is known concerning fungal or plant genetic factors that impact seed transmission and mechanisms underlying this key biological trait have yet to be clarified. Such lack of available data could be probably explained by the absence of suitable model pathosystem to study plant-fungus interactions during the plant reproductive phase. Results: Here we report on setting up a new pathosystem that could facilitate the study of fungal seed transmission. Reproductive organs of Arabidopsis thaliana were inoculated with Alternaria brassicicola conidia. Parameters that could influence the seed transmission efficiency were tested to define optimal seed infection conditions. Microscopic observations revealed that the fungus enters siliques through cellular junctions, replum and stomata, and into seed coats either directly or through cracks. The ability of the fungal mutant nik1delta3 to transmit to A. thaliana seeds was analyzed. A significant decrease in seed transmission rate was observed compared to the wild-type parental strain, confirming that a functional osmoregulation pathway is required for efficient seed transmission of the fungus. Similarly, to test the role of flavonoids in seed coat protection against pathogens, a transparent testa Arabidopsis mutant (tt4-1) not producing any flavonoid was used as host plant. Unexpectedly, tt4-1 seeds were infected to a significantly lower extent than wild-type seeds, possibly due to over-accumulation of other antimicrobial metabolites. Conclusions: The Arabidopsis thaliana - Alternaria brassicicola pathosystem, that have been widely used to study plant-pathogen interactions during the vegetative phase, also proved to constitute a suitable model pathosystem for detailed analysis of plant-pathogen interactions during the reproductive phase. We demonstrated that it provides an excellent system for investigating the impact of different fungal or plant mutations on the seed transmission process and therefore paves the way towards future high-throughput screening of both Arabidopsis and fungal mutant. http://www.plantmethods.com/content/8/1/16 Stephanie Pochon Emmanuel Terrasson Thomas Guillemette Beatrice Iacomi-Vasilescu Sonia Georgeault Marjorie Juchaux Romain Berruyer Isabelle Debeaujon Philippe Simoneau Claire Campion Plant Methods 2012, null:16 2012-05-09T00:00:00Z doi:10.1186/1746-4811-8-16 Fungal seed transmission /content/figures/1746-4811-8-16-toc.gif Plant Methods 1746-4811 ${item.volume} 16 2012-05-09T00:00:00Z PDF Genetic resources available for Arabidopsis thaliana make this species particularly attractive as a model for molecular genetic studies of guard cell homeostasis, transport and signalling, but this facility is not matched by accessible tools for quantitative analysis of transport in the intact cell. We have developed a reliable set of procedures for voltage clamp analysis of guard cells from Arabidopsis leaves. These procedures greatly simplify electrophysiological recordings, extending the duration of measurements and scope for analysis of the predominant K+ and anion channels to that achieved previously in work with Vicia and tobacco guard cells. http://www.plantmethods.com/content/8/1/15 Zhong-Hua Chen Cornelia Eisenach Xin-Qin Xu Adrian Hills Michael Blatt Plant Methods 2012, null:15 2012-05-06T00:00:00Z doi:10.1186/1746-4811-8-15 Guard cells are functionally important in their role regulating stomatal aperture and offer many advantages for electrophysiological analysis. The detailed protocols for voltage clamp analysis of Arabidopsis guard cells set out here allow measurements of much longer duration than was previously possible. /content/figures/1746-4811-8-15-toc.gif Plant Methods 1746-4811 ${item.volume} 15 2012-05-06T00:00:00Z PDF 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 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 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: Accurate quantification of xylem sap ABA concentrations is important to underpin models of root-to-shoot ABA signalling to predict the physiological effects of soil drying. Growing tomato plants in a whole plant pressure chamber allowed sequential xylem sap collection from a detached leaf, the petiole stub of an otherwise intact plant and finally the de-topped root system of the same plant, to determine the impact of xylem sap sampling methodology on xylem ABA concentration. Since xylem sap can contain bound forms of ABA, a novel gas chromatography-mass spectrometry (GC-MS) procedure was developed to chemically separate free ABA from two in planta bound ABA forms known as Adducts I and II and ABA-glucose-ester (ABA-GE). Results: Xylem sap ABA concentrations were highly dependent on the sampling methodology used: the highest concentrations were detected in sap collected by applying an overpressure to detached leaves following the measurement of leaf water potential. Irrespective of xylem sap source, the wild-type cultivars Ailsa Craig and Rheinlands Ruhm had higher free ABA concentrations than a range of ABA-deficient mutants (notabilis, flacca and sitiens). However, in the mutants, concentrations of bound forms of ABA were similar to wild-type plants, and similar to free ABA concentrations. Conclusions: Although xylem concentrations of these bound ABA forms and ABA-GE suggest they have a limited physiological impact on ABA homeostasis in tomato, the methods developed here will allow a more complete understanding of ABA biochemistry and root-to-shoot signalling in species known to have higher concentrations of these compounds. http://www.plantmethods.com/content/8/1/11 Andrew Netting Julian Theobald Ian Dodd Plant Methods 2012, null:11 2012-03-22T00:00:00Z doi:10.1186/1746-4811-8-11 The authors describe improved methodology for collecting xylem sap and a novel GC-MS procedure for quantifying the presence of free and bound forms of abscisic acid (ABA). Although the abundance of these bound forms of ABA in tomato is found to be low, the technique will be of particular relevance for those species where bound forms are known to represent a much more significant fraction of the total xylem sap ABA content. /content/figures/1746-4811-8-11-toc.gif Plant Methods 1746-4811 ${item.volume} 11 2012-03-22T00:00:00Z XML Background: Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism. Results: Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A) gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP) reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants. Conclusions: Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes. http://www.plantmethods.com/content/8/1/10 Sachiko Arase Megumi Kasai Akira Kanazawa Plant Methods 2012, null:10 2012-03-19T00:00:00Z doi:10.1186/1746-4811-8-10 /content/figures/1746-4811-8-10-toc.gif Plant Methods 1746-4811 ${item.volume} 10 2012-03-19T00:00:00Z PDF Background: Temporal and tissue-specific patterns of gene expression play important roles in functionality of a biological system. Real-time quantitative polymerase chain reaction (qPCR) technique has been widely applied to single gene expressions, but its potential has not been fully released as most results have been obtained as fold changes relative to control conditions. Absolute quantification of transcripts as an alternative method has yet to gain popularity because of unresolved issues. Results: We propose a solution here with a novel procedure, which may accurately quantify the total cDNA conventionally prepared from a biological sample at the resolution of ~70 pg/μl, and reliably estimate the absolute numbers of transcripts in a picogram of cDNA. In comparison to the relative quantification, cDNA-based absolute (CBA) qPCR method is found to be more sensitive to gene expression variations caused by factors such as developmental and environmental variations. If the number of target transcript copies is further normalized by reference transcripts, cell-level variation pattern of the target gene expression may also be detectable during a developmental process, as observed here in cases across species (Ipomoea purpurea, Nicotiana benthamiana) and tissues (petals and leaves). Conclusion: By allowing direct comparisons of results across experiments, the new procedure opens a window to make inferences of gene expression patterns across a broad spectrum of living systems and tissues. Such comparisons are urgently needed for biological interpretations of gene expression variations in diverse cells. http://www.plantmethods.com/content/8/1/9 Yingqing Lu Lulu Xie Jiani Chen Plant Methods 2012, null:9 2012-03-09T00:00:00Z doi:10.1186/1746-4811-8-9 Absolute rather than relative quantification of mRNAs Standard methods for quantifying gene transcripts yield relative rather than absolute data (e.g. fold change compared to control treatments). The authors describe a real-time PCR method that enables absolute quantification of cDNAs. The objective is to allow the comparison of gene expression between different tissues, developmental stages, and even different experiments. /content/figures/1746-4811-8-9-toc.gif Plant Methods 1746-4811 ${item.volume} 9 2012-03-09T00:00:00Z XML Three terrestrial plants are known to perform C4 photosynthesis without the dual-cell system by partitioning two distinct types of chloroplasts in separate cytoplasmic compartments. We report herein a protocol for isolating the dimorphic chloroplasts from Bienertia sinuspersici. Hypo-osmotically lysed protoplasts under our defined conditions released intact compartments containing the central chloroplasts and intact vacuoles with adhering peripheral chloroplasts. Following Percoll step gradient purification both chloroplast preparations demonstrated high homogeneities as evaluated from the relative abundance of respective protein markers. This protocol will open novel research directions toward understanding the mechanism of single-cell C4 photosynthesis. http://www.plantmethods.com/content/8/1/8 Shiu-Cheung Lung Makoto Yanagisawa Simon Chuong Plant Methods 2012, null:8 2012-03-06T00:00:00Z doi:10.1186/1746-4811-8-8 Facilitating studies of a novel type of C4 photosynthesis C4-type photosynthesis is of major scientific interest because of its greater efficiency compared to the more common C3-type. Bienertia sinuspersici belongs to a select group of just three terrestrial higher plant species known to perform C4 photosynthesis without the segregation of different parts of the pathway into different cell types. It achieves this feat by partitioning its photosynthetic functions between two distinct types of chloroplast that are located in separate cytoplasmic compartments of the same cell. The authors describe methodology for isolating the two types of chloroplast, providing a means to investigate the biochemistry and physiology that underlies this intriguing photosynthetic system. /content/figures/1746-4811-8-8-toc.gif Plant Methods 1746-4811 ${item.volume} 8 2012-03-06T00:00:00Z XML