NEWS FROM
PHYSIOLOGIA PLANTARUM
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Published monthly on behalf of SPPS by Wiley-Blackwell.
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Arabidopsis get excited
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Action potentials, i.e. rapid and transient changes of the membrane potential that travel over long distances, are not unique to animals. Several plants exploit them for various purposes: capturing insects in the carnivorous Venus flytrap, rapid movement of leaves in Mimosa and triggering of a systemic response following injure in tomato. However, action potentials in plants have not been thoroughly studied due to the lack of a suitable and reproducible model system. Now Swiss scientists propose Arabidopsis thaliana as such a model. Excitation by electrodes in the distal part of the leaf caused reproducible action potentials that travelled down through the petiole at a speed of 1.2 mm/s.
Read full article free: Favre & Agosti (October 2007) Physiologia Plantarum 131: 263-272
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NEWS IN BRIEF
FROM OTHER JOURNALS
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Potatoes highlight the plant-fungi relationship
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Source: Drissner et al (12 October 2007) Science 318: 265-268
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Recipe for better and sustainable rice
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Source: Zhang (1 October 2007) PNAS doi:10.1073/pnas.0708013104
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Bioimaging - a coloured revolution
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Above: The fluorescent probe (black sphere) absorb a photon (blue) and moves to an exited state. When it returns to the stable ground state a photon (green) is released. In some cases, two low-energy photons (red) can excite a probe which subsequently emits a single high-energy photon (green). Below: Typical excitation and emission spectra for a fluorescent probe. From Vonesh et al (May 2006) IEEE Sig Proc Mag
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Biologists have come to depend more and more on bioimaging as a tool to identify and locate specific proteins and molecules in their natural environment. With the recent development of probes and microscopes, biological processes can be monitored in real time in two or three dimensions. And observations can even be made non-invasively so a cellular process can be followed over an extended period of time.
Bioimaging relies on probes that are attached to proteins or other molecules of interest. These probes are fluorescent and as such emit light of a specific wavelength when they are excited by light of another - usually shorter - wavelength. Cells and their components are mainly transparent and the myriad of molecules within them are indistinguishable from each other in a normal microscope. But if the protein of interest lights up in bright green, it is easy to detect and distinguish from all other molecules in the cell.
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A GFP-tagged thaumatin-like protein is expressed in the cell walls of a trichome. From Runions (2003) Nature Cell Biology 5:699
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Bioimaging had its breakthrough in 1994 when Martin Chalfie from Colombia University in the US expressed a protein from a jellyfish in E. coli and the round worm C. elegans. The protein was GFP (Green Fluorescent Protein) which is fluorescent and emits green light when excited by blue light. The result was overwhelming with bright green bacteria and worms, but the real power of bioimaging was demonstrated when scientists realized that GFP by means of genetic engineering can be fused to natural proteins in animals, plants or microorganisms. Now, not the whole organism but rather a particular protein will light up, so its distribution within the cell is visualized. Moreover, GFP was fused to promoters so it could tell where and when expression was directed.
The art of bioimaging is to develop probes that can be specifically attached to certain molecules and that emit light in distinct colours so that several probes can be used in the same sample in order to monitor more than one biological process at a time. GFP has been modified to emit cyan, blue and yellow light and other proteins adds red to the palette. A classical method to label proteins or other molecules is to raise antibodies against them and couple these to a fluorescent probe. Due to the size of the antibodies this method will, however, will often render the molecule of interest biologically inactive and is accordingly not normally compatible with life processes. Also a large number of rather simple organic molecules have been developed as fluorescent probes. These can be injected directly into cells or tissue and depending on their nature they will bind to certain proteins or molecules. One of these is DAPI which specifically binds DNA and emits blue light.
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Fluorescent quantum dots can simultaneously reveal several structures of the cell in distinct colours. Blue = nucleus; pink = a nucleic protein; yellow = mitochondria; green = microtubules; red = actin filaments. Image by Quantum Dot Corp.
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A new type of fluorescent probes is the quantum dots or qdots. They are inorganic semiconductor nanocrystals, and their fluorescent characteristics depends largely on their size. The general rule is that with increasing size of the crystal, the wavelength of the emitted light becomes longer, i.e. more reddish. By synthesizing them in different colours it is possible to design a wide range of probes in different colours. Furthermore, it is relatively easy to design peptides where one end that bind the qdots and the other has some kind of functionality, e.g. binding specifically to a molecule of interest. In this way it is possible to simultaneously study several processes within a cell or tissue.
Irrespective of the probe used, it must be viewed through a fluorescence microscope in order to bee visualized. This is basically an ordinary microscope but it has a built-in light source that produce light at a particular wavelength that will excite the probe being used. The classical widefield fluorescence microscope broadly illuminates the sample and emitted light from the whole field of sight is reflected to the microscopes eyepiece, where it can be detected by the naked eye or a digital camera. The main downside of this straight forward method is that the excitation light is difficult to focus so emission will occur from out-of-focus points, leading to somewhat blurred images with limited resolution.
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The confocal laser scanning microscope uses a laser beam for excitation of a narrow point in the sample. From Vonesh et al (May 2006) IEEE Sig Proc Mag
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This problem is solved in the confocal laser scanning microscope (CLSM) where a focused laser beam directs the excitation light to a narrow point in not only the horizontal but also in the vertical plane. Only markers at this very location will now fluoresce and the relatively weak emission light is sent to a photo multiplier instead of the naked eye. The information is stored in a computer and by means of a mirror the laser beam is moved slightly. In this way, the whole sample is progressively scanned and the computer generates a complete picture all the information. The laser can penetrate he tissue, so by adjusting focus to a deeper layer the sample might be successively rescanned in several planes, allowing for generation of a 3D representation of the sample.
Apart from giving beautiful spatial pictures that can be turned around on the screen in order to see the sample from all sides, the confocal laser scanning microscope is ideal for studying the interior of living organisms. A leaf can be studied while still attached to the plant with its roots in the soil, and brain processes have been studied by bioimaging in live rats.
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FRET combines two fluorescent probes that interact in the presence of a specific molecule. From http://probes.invitrogen.com/products/premo/
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The sophisticated CLSM microscope can be combined with just as sophisticated probes whose fluorescence depends on their chemical surroundings. Some probes are sensitive to ions like calcium or zinc, while others might respond to changes in the pH. A special technique called FRET (fluorescence resonance energy transfer) is often used to sense changes in the Ca2+ concentration by attaching two different probes, e.g. CFP and YFP, on each end of a calcium binding calmodulin peptide. In the absence of Ca2+, beaming with a blue laser will only excite CFP leading to emission of cyan light. In the presence of Ca2+, however, the peptide folds and bring the two probes in close proximity so they become one functional probe. Instead of emitting cyan light, CFP will directly transfer all its energy to YFP that in turn emit yellow light. Accordingly, a shift from low to high Ca2+ concentration can be observed as a shift from cyan to yellow fluorescence.
Another technique called FRAP (fluorescence recovery after photobleaching) are used to follow the diffusion and mobility of the probe in order to study e.g. phloem transport. High-intensity light is used to intentionally bleach a small region of the sample, thereby rendering it non-fluorescent. As other probes from the surroundings flow back into the region it regains fluorescence, yielding information about the mobility of the probe. Yet another method, FLIM (fluorescence lifetime image microscopy), is used both to discriminate between multiple probes with similar emission spectra and to detect chemical changes in the local environment. It relies on the fact, that the fluorescence of every probe has a unique lifetime and that it depends on certain environmental factors. By using a picosecond pulsed laser for excitation and recording the arrival time of the emitted photons with a high-speed photodetector the fluorescence lifetime can be measured.
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Bioimaging is among the Future Technologies for Life that University of Copenhagen will focus on. Courtesy Alexander Schulz
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In Denmark, bioimaging is used extensively several places and among plant biologists in particular by Professor Alexander Schulz at the University of Copenhagen. His group focuses on the cell biology of symplasmic communication in higher plants and bioimaging techniques like FRAP and 2-photon laser scanning microscopy are used to visualize the transport processes. Alexander Schulz is heading a new initiative called Future Technologies for Life that will eventually combine all the bioimaging expertises at four faculties of the university.
You can read more about bioimaging on Alexander Schulz's homepage.
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