Plant Methods (PLANT METHODS)

Publications in this journal

• Article: An efficient flat-surface collar-free grafting method for Arabidopsis thaliana seedlings.

  Nayelli Marsch Martínez, John Franken, Karla L Gonzalez-Aguilera, Stefan de Folter, Gerco Angenent, Elena R Alvarez-Buylla
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  ABSTRACT: BACKGROUND: Grafting procedures are an excellent tool to study long range signalling processes within a plant. In the last decade, suitable flat-surface grafting procedures for young Arabidopsis seedlings using a collar to support the graft have been developed, allowing the study of long-range signals from a molecular perspective. RESULTS: In the modification presented here, scion and stock are put together on the medium without supporting elements, while cotyledons are removed from the scion, resulting in increased grafting success that can reach up to 100%. At the same time, the protocol enables to process as many as 36 seedlings per hour, which combined with the high success percentage represents increased efficiency per time unit. CONCLUSIONS: Growing cotyledons usually push the scion and the rootstock away in the absence of a supporting element. Removing them at the grafting step greatly improved success rate and reduced post-grafting manipulations.
  Plant Methods 05/2013; 9(1):14.
• Article: A selective pretreatment method for determination of endogenous active brassinosteroids in plant tissues: double layered solid phase extraction combined with boronate affinity polymer monolith microextraction.

  Jun Ding, Li-Jing Mao, Bi-Feng Yuan, Yu-Qi Feng
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  ABSTRACT: BACKGROUND: Brassinosteriods (BRs), a group of important phytohormones, have various effects on plant growth and development. However, their physiological functions in plants have not been fully understood to date. Endogenous BRs in plant tissue are extremely low and the elucidation of BRs functions relies on sensitive detection method. Reported methods for the determination of BRs required large amount of plant tissue, tedious pretreatment process, and were lack of selectivity. Therefore, development of a simple and selective method for the sensitive quantification of BRs is highly needed. RESULTS: We established a pretreatment method of BRs in plant tissues by employing double layered solid phase extraction (DL/SPE) combined with boronate affinity polymer monolith microextraction (BA/PMME). After the initial depigmentation with DL/SPE cartridge, boronate affinity polymer BA/PMME was employed to selectively extract BRs from sample matrix. Uniquely, most sample matrix was successfully removed by BA monolith purification. Using this method, BRs was determined by liquid chromatography-mass spectrometry (LC-MS). Endogenous active BRs could be detected in only 1 g fresh weigh (FW) leaves or 0.5 g FW flower tissues. CONCLUSION: A DL/SPE-BA/PMME pretreatment method for the determination of endogenous brassinosteroids in plant tissues was developed and validated. The proposed method was sensitive and selective. The proposed method may be further developed for the determination of other BRs including their precursors and conjugates.
  Plant Methods 04/2013; 9(1):13.
• Article: Using an ensemble of statistical metrics to quantify large sets of plant transcription factor binding sites.

  Parsa Hosseini, Ivan Ovcharenko, Benjamin F Matthews
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  ABSTRACT: Background From initial seed germination through reproduction, plants continuously reprogram their transcriptional repertoire to facilitate growth and development. This dynamic is mediated by a diverse but inextricably-linked catalog of regulatory proteins called transcription factors (TFs). Statistically quantifying TF binding site (TFBS) abundance in promoters of differentially expressed genes can be used to identify binding site patterns in promoters that are closely related to stress-response. Output from today's transcriptomic assays necessitates statistically-oriented software to handle large promoter-sequence sets in a computationally tractable fashion. Results We present Marina, an open-source software for identifying over-represented TFBSs from amongst large sets of promoter sequences, using an ensemble of 7 statistical metrics and binding-site profiles. Through software comparison, we show that Marina can identify considerably more over-represented plant TFBSs compared to a popular software alternative. Conclusions Marina was used to identify over-represented TFBSs in a two time-point RNA-Seq study exploring the transcriptomic interplay between soybean (Glycine max) and soybean rust (Phakopsora pachyrhizi). Marina identified numerous abundant TFBSs recognized by transcription factors that are associated with defense-response such as WRKY, HY5 and MYB2. Comparing results from Marina to that of a popular software alternative suggests that regardless of the number of promoter-sequences, Marina is able to identify significantly more over-represented TFBSs.
  Plant Methods 04/2013; 9(1):12.
• Article: Visualizing water-filled versus embolized status of xylem conduits by desktop x-ray microtomography.

  Jussi-Petteri Suuronen, Marko Peura, Kurt Fagerstedt, Ritva Serimaa
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  ABSTRACT: BACKGROUND: The hydraulic conductivity of the stem is a major factor limiting the capability of trees to transport water from the soil to transpiring leaves. During drought conditions, the conducting capacity of xylem can be reduced by some conduits being filled with gas, i.e. embolized. In order to understand the dynamics of embolism formation and repair, considerable attention has been given to developing reliable and accurate methods for quantifying the phenomenon. In the past decade, non-destructive imaging of embolism formation in living plants has become possible. Magnetic resonance imaging has been used to visualize the distribution of water within the stem, but in most cases it is not possible to resolve individual cells. Recently, high-resolution synchrotron x-ray microtomography has been introduced as a tool to visualize the water contents of individual cells in vivo, providing unprecedented insight into the dynamics of embolism repair. We have investigated the potential of an x-ray tube -based microtomography setup to visualize and quantify xylem embolism and embolism repair in water-stressed young saplings and shoot tips of Silver and Curly birch (Betula pendula and B. pendula var. carelica). RESULTS: From the microtomography images, the water-filled versus gas-filled status of individual xylem conduits can be seen, and the proportion of stem cross-section that consists of embolized tissue can be calculated. Measuring the number of embolized vessels in the imaged area is a simple counting experiment. In the samples investigated, wood fibers were cavitated in a large proportion of the xylem cross-section shortly after watering of the plant was stopped, but the number of embolized vessels remained low several days into a drought period. Under conditions of low evaporative demand, also refilling of previously embolized conduits was observed. CONCLUSIONS: Desktop x-ray microtomography is shown to be an effective method for evaluating the water-filled versus embolized status of the stem xylem in a small living sapling. Due to its non-destructive nature, the risk of inducing embolisms during sampling is greatly reduced. Compared with synchrotron imaging beamlines, desktop microtomography offers easier accessibility, while maintaining sufficient resolution to visualize the water contents of individual cells.
  Plant Methods 04/2013; 9(1):11.
• Article: Emission spectra profiling of fluorescent proteins in living plant cells.

  Evelien Mylle, Mirela-Corina Codreanu, Joanna Boruc, Eugenia Russinova
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  ABSTRACT: BACKGROUND: Fluorescence imaging at high spectral resolution allows the simultaneous recording of multiple fluorophores without switching optical filters, which is especially useful for time-lapse analysis of living cells. The collected emission spectra can be used to distinguish fluorophores by a computation analysis called linear unmixing. The availability of accurate reference spectra for different fluorophores is crucial for this type of analysis. The reference spectra used by plant cell biologists are in most cases derived from the analysis of fluorescent proteins in solution or produced in animal cells, although these spectra are influenced by both the cellular environment and the components of the optical system. For instance, plant cells contain various autofluorescent compounds, such as cell wall polymers and chlorophyll, that affect the spectral detection of some fluorophores. Therefore, it is important to acquire both reference and experimental spectra under the same biological conditions and through the same imaging systems. RESULTS: Entry clones (pENTR) of fluorescent proteins (FPs) were constructed in order to create C- or N-terminal protein fusions with the MultiSite Gateway recombination technology. The emission spectra for eight FPs, fused C-terminally to the A- or B-type cyclin dependent kinases (CDKA;1 and CDKB1;1) and transiently expressed in epidermal cells of tobacco (Nicotiana benthamiana), were determined by using the Olympus FluoViewTM FV1000 Confocal Laser Scanning Microscope. These experimental spectra were then used in unmixing experiments in order to separate the emission of fluorophores with overlapping spectral properties in living plant cells. CONCLUSIONS: Spectral imaging and linear unmixing have a great potential for efficient multicolor detection in living plant cells. The emission spectra for eight of the most commonly used FPs were obtained in epidermal cells of tobacco leaves and used in unmixing experiments. The generated set of FP Gateway entry vectors represents a valuable resource for plant cell biologists.
  Plant Methods 04/2013; 9(1):10.
• Article: Rapid separation of developing Arabidopsis seeds from siliques for RNA or metabolite analysis.

  Philip David Bates, Jeremy Burke Jewell, John Browse
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  ABSTRACT: BACKGROUND: Protein, starch and oil produced in plant seeds are major renewable sources of food, chemicals and biofuels. Developing Arabidopsis thaliana seeds are commonly utilized as a model for seed crop research. However, due to the very small size of Arabidopsis seeds efficient collection of large amounts of tissue for gene expression or metabolite analysis is very difficult and time consuming.Results/conclusions: Here we describe a method that allows very rapid separation and collection of large amounts of developing Arabidopsis seeds from their encapsulating silique tissue after flash freezing whole siliques in liquid nitrogen. The efficient popping open of the frozen siliques on dry ice and filtering the seeds away from the silique tissue with liquid nitrogen cooled funnels and sieves allows large amounts of developing seeds to be quickly isolated while remaining frozen. This method increases the speed of developing seed collection approximately 10 fold over methods which dissect individual siliques one at a time.
  Plant Methods 03/2013; 9(1):9.
• Article: Recovering complete plant root system architectures from soil via X-ray mu-Computed Tomography.

  Stefan Mairhofer, Susan Zappala, Saoirse Tracy, Craig Sturrock, Malcolm John Bennett, Sacha Jon Mooney, Tony Paul Pridmore
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  ABSTRACT: BACKGROUND: X-ray micro-Computed Tomography (muCT) offers the ability to visualise the three-dimensional structure of plant roots growing in their natural environment -- soil. Recovery of root architecture descriptions from X-ray CT data is, however, challenging. The X-ray attenuation values of roots and soil overlap, and the attenuation values of root material vary. Any successful root identification method must both explicitly target root material and be able to adapt to local changes in root properties.RooTrak meets these requirements by combining the level set method with a visual tracking framework and has been shown to be capable of segmenting a variety of plant roots from soil in X-ray muCT images. The approach provides high quality root descriptions, but tracks root systems top to bottom and so omits upward-growing (plagiotropic) branches. RESULTS: We present an extension to RooTrak which allows it to extract plagiotropic roots. An additional backward-looking step revisits the previous image, marking possible upward-growing roots. These are then tracked, leading to efficient and more complete recovery of the root system. Results show clear improvement in root extraction, without which key architectural traits would be underestimated. CONCLUSIONS: The visual tracking framework adopted in RooTrak provides the focus and flexibility needed to separate roots from soil in X-ray CT imagery and can be extended to detect plagiotropic roots. The extended software tool produces more complete descriptions of plant root structure and supports more accurate computation of architectural traits.
  Plant Methods 03/2013; 9(1):8.
• Article: Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants.

  Simon J Conn, Bradleigh Hocking, Maclin Dayod, Bo Xu, Asmini Athman, Sam Henderson, Lucy Aukett, Vanessa Conn, Monique K Shearer, Sigfredo Fuentes, Stephen D Tyerman, Matthew Gilliham
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  ABSTRACT: BACKGROUND: Hydroponic growth systems are a convenient platform for studying whole plant physiology. However, we found through trialling systems as they are described in the literature that our experiments were frequently confounded by factors that affected plant growth, including algal contamination and hypoxia. We also found the way in which the plants were grown made them poorly amenable to a number of common physiological assays. RESULTS: The drivers for the development of this hydroponic system were: 1) the exclusion of light from the growth solution; 2) to simplify the handling of individual plants, and 3) the growth of the plant to allow easy implementation of multiple assays. These aims were all met by the use of pierced lids of black microcentrifuge tubes. Seed was germinated on a lid filled with an agar-containing germination media immersed in the same solution. Following germination, the liquid growth media was exchanged with the experimental solution, and 14-21 days seedlings were transferred to larger tanks with aerated solution where they remained until experimentation. We provide details of the protocol including composition of the basal growth solution, and separate solutions with altered calcium, magnesium, potassium or sodium supply whilst maintaining the activity of the majority of other ions. We demonstrate the adaptability of this system for: gas exchange measurement on single leaves and whole plants; qRT-PCR to probe the transcriptional response of roots or shoots to altered nutrient composition in the growth solution (we demonstrate this using high and low calcium supply); producing highly competent mesophyll protoplasts; and, accelerating the screening of Arabidopsis transformants. This system is also ideal for manipulating plants for micropipette techniques such as electrophysiology or SiCSA. CONCLUSIONS: We present an optimised plant hydroponic culture system that can be quickly and cheaply constructed, and produces plants with similar growth kinetics to soil-grown plants, but with the advantage of being a versatile platform for a myriad of physiological and molecular biological measurements on all plant tissues at all developmental stages. We present 'tips and tricks' for the easy adoption of this hydroponic culture system.
  Plant Methods 02/2013; 9(1):4.
• Article: Rhizo-lysimetry: facilities for the simultaneous study of root behaviour and resource use by agricultural crop and pasture systems.

  Philip L Eberbach, Jeffrey Hoffmann, Sergio J Moroni, Leonard J Wade, Leslie A Weston
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  ABSTRACT: BACKGROUND: Rhizo-lysimeters offer unique advantages for the study of plants and their interactions with soils. In this paper, an existing facility at Charles Sturt University in Wagga Wagga Australia is described in detail and its potential to conduct both ecophysiological and ecohydrological research in the study of root interactions of agricultural crops and pastures is quantitatively assessed. This is of significance to future crop research efforts in southern Australia, in light of recent significant long-term drought events, as well as potential impacts of climate change as predicted for the region. The rhizo-lysimeter root research facility has recently been expanded to accommodate larger research projects over multiple years and cropping rotations. RESULTS: Lucerne, a widely-grown perennial pasture in southern Australia, developed an expansive root system to a depth of 0.9 m over a twelve month period. Its deeper roots particularly at 2.05 m continued to expand for the duration of the experiment. In succeeding experiments, canola, a commonly grown annual crop, developed a more extensive (approximately 300%) root system than wheat, but exhibited a slower rate of root elongation at rates of 7.47 x 10-3 m day-1 for canola and 1.04 x10-2 m day-1 for wheat. A time domain reflectometry (TDR) network was designed to accurately assess changes in soil water content, and could assess water content change to within 5% of the amount of water applied. CONCLUSIONS: The rhizo-lysimetry system provided robust estimates of root growth and soil water change under conditions representative of a field setting. This is currently one of a very limited number of global research facilities able to perform experimentation under field conditions and is the largest root research experimental laboratory in the southern hemisphere.
  Plant Methods 01/2013; 9(1):3.
• Article: Synchronous high-resolution phenotyping of leaf and root growth in Nicotiana tabacum over 24-h periods with GROWMAP-plant.

  Tom Ruts, Shizue Matsubara, Achim Walter
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  ABSTRACT: BACKGROUND: Root growth is highly responsive to temporal changes in the environment. On the contrary, diel (24 h) leaf expansion in dicot plants is governed by endogenous control and therefore its temporal pattern does not strictly follow diel changes in the environment. Nevertheless, root and shoot are connected with each other through resource partitioning and changing environments for one organ could affect growth of the other organ, and hence overall plant growth. RESULTS: We developed a new technique, GROWMAP-plant, to monitor growth processes synchronously in leaf and root of the same plant with a high resolution over the diel period. This allowed us to quantify treatment effects on the growth rates of the treated and non-treated organ and the possible interaction between them. We subjected the root system of Nicotiana tabacum seedlings to three different conditions: constant darkness at 22[degree sign]C (control), constant darkness at 10[degree sign]C (root cooling), and 12 h/12 h light--dark cycles at 22[degree sign]C (root illumination). In all treatments the shoot was kept under the same 12 h/12 h light--dark cycles at 22[degree sign]C. Root growth rates were found to be constant when the root-zone environment was kept constant, although the root cooling treatment significantly reduced root growth. Root velocity was decreased after light-on and light-off events of the root illumination treatment, resulting in diel root growth rhythmicity. Despite these changes in root growth, leaf growth was not affected substantially by the root-zone treatments, persistently showing up to three times higher nocturnal growth than diurnal growth. CONCLUSION: GROWMAP-plant allows detailed synchronous growth phenotyping of leaf and root in the same plant. Root growth was very responsive to the root cooling and root illumination, while these treatments altered neither relative growth rate nor diel growth pattern in the seedling leaf. Our results that were obtained simultaneously in growing leaves and roots of the same plants corroborate the high sensitivity of root growth to the environment and the contrasting robustness of diel growth patterns in dicot leaves. Further, they also underpin the importance to carefully control the experimental conditions for root growth analysis to avoid or/and minimize artificial complications.
  Plant Methods 01/2013; 9(1):2.
• Article: Novel scanning procedure enabling the vectorization of entire rhizotron-grown root systems.

  Guillaume Lobet, Xavier Draye
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  ABSTRACT: This paper presents an original spit-and-combine imaging procedure that enables the complete vectorization of complex root systems grown in rhizotrons. The general principle of the method is to (1) separate the root system into a small number of large pieces to reduce root overlap, (2) scan these pieces one by one, (3) analyze separate images with a root tracing software and (4) combine all tracings into a single vectorized root system. This method generates a rich dataset containing morphological, topological and geometrical information of entire root systems grown in rhizotrons. The utility of the method is illustrated with a detailed architectural analysis of a 20-day old maize root system, coupled with a spatial analysis of water uptake patterns.
  Plant Methods 01/2013; 9(1):1.
• Article: Inflorescence stem grafting made easy in Arabidopsis.

  Nazia Nisar, Shelley Verma, Barry J Pogson, Christopher I Cazzonelli
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  ABSTRACT: BACKGROUND: Plant grafting techniques have deepened our understanding of the signals facilitating communication between the root and shoot, as well as between shoot and reproductive organs. Transmissible signalling molecules can include hormones, peptides, proteins and metabolites: some of which travel long distances to communicate stress, nutrient status, disease and developmental events. While hypocotyl micrografting techniques have been successfully established for Arabidopsis to explore root to shoot communications, inflorescence grafting in Arabidopsis has not been exploited to the same extent. Two different strategies (horizontal and wedge-style inflorescence grafting) have been developed to explore long distance signalling between the shoot and reproductive organs. We developed a robust wedge-cleft grafting method, with success rates greater than 87%, by developing better tissue contact between the stems from the inflorescence scion and rootstock. We describe how to perform a successful inflorescence stem graft that allows for reproducible translocation experiments into the physiological, developmental and molecular aspects of long distance signalling events that promote reproduction. RESULTS: Wedge grafts of the Arabidopsis inflorescence stem were supported with silicone tubing and further sealed with parafilm to maintain the vascular flow of nutrients to the shoot and reproductive tissues. Nearly all (87%) grafted plants formed a strong union between the scion and rootstock. The success of grafting was scored using an inflorescence growth assay based upon the growth of primary stem. Repeated pruning produced new cauline tissues, healthy flowers and reproductive siliques, which indicates a healthy flow of nutrients from the rootstock. Removal of the silicone tubing showed a tightly fused wedge graft junction with callus proliferation. Histological staining of sections through the graft junction demonstrated the differentiation of newly formed vascular connections, parenchyma tissue and lignin accumulation, supporting the presumed success of the graft union between two sections of the primary inflorescence stem. CONCLUSIONS: We describe a simple and reliable method for grafting sections of an Arabidopsis inflorescence stem. This step-by-step protocol facilitates laboratories without grafting experience to further explore the molecular and chemical signalling which coordinates communications between the shoot and reproductive tissues.
  Plant Methods 12/2012; 8(1):50.
• Article: Protocol: Chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species.

  Xin Lin, Leïla Tirichine, Chris Bowler
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  ABSTRACT: In this report we describe a chromatin immunoprecipitation (ChIP) protocol for two fully sequenced model diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana. This protocol allows the extraction of satisfactory amounts of chromatin and gives reproducible results. We coupled the ChIP assay with real time quantitative PCR. Our results reveal that the two major histone marks H3K4me2 and H3K9me2 exist in P. tricornutum and T. pseudonana. As in other eukaryotes, H3K4me2 marks active genes whereas H3K9me2 marks transcriptionally inactive transposable elements. Unexpectedly however, T. pseudonana housekeeping genes also show a relative enrichment of H3K9me2. We also discuss optimization of the procedure, including growth conditions, cross linking and sonication. Validation of the protocol provides a set of genes and transposable elements that can be used as controls for studies using ChIP in each diatom species. This protocol can be easily adapted to other diatoms and eukaryotic phytoplankton species for genetic and biochemical studies.
  Plant Methods 12/2012; 8(1):48.
• Article: Plant lighting system with five wavelength-band light-emitting diodes providing photon flux density and mixing ratio control.

  Akira Yano, Kazuhiro Fujiwara
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  ABSTRACT: BACKGROUND: Plant growth and development depend on the availability of light. Lighting systems therefore play crucial roles in plant studies. Recent advancements of light-emitting diode (LED) technologies provide abundant opportunities to study various plant light responses. The LED merits include solidity, longevity, small element volume, radiant flux controllability, and monochromaticity. To apply these merits in plant light response studies, a lighting system must provide precisely controlled light spectra that are useful for inducing various plant responses. RESULTS: We have developed a plant lighting system that irradiated a 0.18 m2 area with a highly uniform distribution of photon flux density (PFD). The average photosynthetic PFD (PPFD) in the irradiated area was 438 micro-mol m-2 s-1 (coefficient of variation 9.6%), which is appropriate for growing leafy vegetables. The irradiated light includes violet, blue, orange-red, red, and far-red wavelength bands created by LEDs of five types. The PFD and mixing ratio of the five wavelength-band lights are controllable using a computer and drive circuits. The phototropic response of oat coleoptiles was investigated to evaluate plant sensitivity to the light control quality of the lighting system. Oat coleoptiles irradiated for 23 h with a uniformly distributed spectral PFD (SPFD) of 1 micro-mol m-2 s-1 nm-1 at every peak wavelength (405, 460, 630, 660, and 735 nm) grew almost straight upwards. When they were irradiated with an SPFD gradient of blue light (460 nm peak wavelength), the coleoptiles showed a phototropic curvature in the direction of the greater SPFD of blue light. The greater SPFD gradient induced the greater curvature of coleoptiles. The relation between the phototropic curvature (deg) and the blue-light SPFD gradient (micro-mol m-2 s-1 nm-1 m-1) was 2 deg per 1 micro-mol m-2 s-1 nm-1 m-1. CONCLUSIONS: The plant lighting system, with a computer with a graphical user interface program, can control the PFD and mixing ratios of five wavelength-band lights. A highly uniform PFD distribution was achieved, although an intentionally distorted PFD gradient was also created. Phototropic responses of oat coleoptiles to the blue light gradient demonstrated the merit of fine controllability of this plant lighting system.
  Plant Methods 11/2012; 8(1):46.
• Article: An UPLC-MS/MS method for highly sensitive high-throughput analysis of phytohormones in plant tissues.

  Gerd Ulrich Balcke, Vinzenz Handrick, Nick Bergau, Mandy Fichtner, Anja Henning, Hagen Stellmach, Alain Tissier, Bettina Hause, Andrej Frolov
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  ABSTRACT: BACKGROUND: Phytohormones are the key metabolites participating in the regulation of multiple functions of plant organism. Among them, jasmonates, as well as abscisic and salicylic acids are responsible for triggering and modulating plant reactions targeted against pathogens and herbivores, as well as resistance to abiotic stress (drought, UV-irradiation and mechanical wounding). These factors induce dramatic changes in phytohormone biosynthesis and transport leading to rapid local and systemic stress responses. Understanding of underlying mechanisms is of principle interest for scientists working in various areas of plant biology. However, highly sensitive, precise and high-throughput methods for quantification of these phytohormones in small samples of plant tissues are still missing. RESULTS: Here we present an LC-MS/MS method for fast and highly sensitive determination of jasmonates, abscisic and salicylic acids. A single-step sample preparation procedure based on mixed-mode solid phase extraction was efficiently combined with essential improvements in mobile phase composition yielding higher efficiency of chromatographic separation and MS-sensitivity. This strategy resulted in dramatic increase in overall sensitivity, allowing successful determination of phytohormones in small (less than 50 mg of fresh weight) tissue samples. The method was completely validated in terms of analyte recovery, sensitivity, linearity and precision. Additionally, it was cross-validated with a well-established GC-MS-based procedure and its applicability to a variety of plant species and organs was verified. CONCLUSION: The method can be applied for the analyses of target phytohormones in small tissue samples obtained from any plant species and/or plant part relying on any commercially available (even less sensitive) tandem mass spectrometry instrumentation.
  Plant Methods 11/2012; 8(1):47.