Marie-Claire Verdus

SIMS study of the calcium-deprivation step related to epidermal meristem production induced in flax by cold shock or radiation from a GSM telephone

ABSTRACT Exposing seedlings of the flax, Linum usitatissimum L., to a variety of weak environmental stresses plus a 2-day calcium deprivation triggers the common response of production of epidermal meristems in the hypocotyl. Here, we show that the same response was induced by a 1 min cold shock. Epidermal meristem production was also induced by a single 2-h exposure to radiation emitted at 0.9 GHz at non-thermal levels by a GSM telephone. This flax-based system is therefore well suited to studying the effects of low intensity stimuli, including those of electromagnetic radiation. To begin to determine the underlying mechanisms, in which calcium is implicated, it is desirable to analyse the changes in ions in the tissues affected. We therefore performed a Secondary Ion Mass Spectrometry (SIMS) study of the distribution of the main inorganic cations in the hypocotyl of control and calcium-deprived seedlings. This showed decreases in calcium, sodium and potassium and an increase in magnesium that did not alter substantially the overall ratio of divalent to monovalent cations.

Hypothesis: Hyperstructures regulate bacterial structure and the cell cycle

A myriad different constituents or elements (genes, proteins, lipids, ions, small molecules etc.) participate in numerous physico-chemical processes to create bacteria that can adapt to their environments to survive, grow and, via the cell cycle, reproduce. We explore the possibility that it is too difficult to explain cell cycle progression in terms of these elements and that an intermediate level of explanation is needed. This level is that of hyperstructures. A hyperstructure is large, has usually one particular function, and contains many elements. Non-equilibrium, or even dissipative, hyperstructures that, for example, assemble to transport and metabolize nutrients may comprise membrane domains of transporters plus cytoplasmic metabolons plus the genes that encode the hyperstructures enzymes. The processes involved in the putative formation of hyperstructures include: metabolite-induced changes to protein affinities that result in metabolon formation, lipid-organizing forces that result in lateral and transverse asymmetries, post-translational modifications, equilibration of water structures that may alter distributions of other molecules, transertion, ion currents, emission of electromagnetic radiation and long range mechanical vibrations. Equilibrium hyperstructures may also exist such as topological arrays of DNA in the form of cholesteric liquid crystals. We present here the beginning of a picture of the bacterial cell in which hyperstructures form to maximize efficiency and in which the properties of hyperstructures drive the cell cycle.

Memory Processes in the Response of Plants to Environmental Signals

Plants are sensitive to stimuli from the environment (e.g. wind, rain, contact, pricking, wounding). They usually respond to such stimuli by metabolic or morphogenetic changes. Sometimes the information corresponding to a stimulus may be “stored” in the plant where it remains inactive until a second stimulus “recalls” this information and finally allows it to take effect. Two experimental systems have proved especially useful in unravelling the main features of these memory-like processes. In the system based on Bidens seedlings, an asymmetrical treatment (e.g. pricking, or gently rubbing one of the seedling cotyledons) causes the cotyledonary buds to grow asymmetrically after release of apical dominance by decapitation of the seedlings. This information may be stored within the seedlings, without taking effect, for at least two weeks; then the information may be recalled by subjecting the seedlings to a second, appropriate, treatment that permits transduction of the signal into the final response (differential growth of the buds). Whilst storage is an irreversible, all-or-nothing process, recall is sensitive to a number of factors, including the intensity of these factors, and can readily be enabled or disabled. In consequence, it is possible to recall the stored message several times successively. In the system based on flax seedlings, stimulation such as manipulation stimulus, drought, wind, cold shock and radiation from a GSM telephone or from a 105 GHz Gunn oscillator, has no apparent effect. If, however, the seedlings are subjected at the same time to transient calcium depletion, numerous epidermal meristems form in their hypocotyls. When the calcium depletion treatment is applied a few days after the mechanical treatment, the time taken for the meristems to appear is increased by a number of days exactly equal to that between the application of the mechanical treatment and the beginning of the calcium depletion treatment. This means that a meristem-production information corresponding to the stimulation treatment has been stored in the plants, without any apparent effect, until the calcium depletion treatment recalls this information to allow it to take effect. Gel electrophoresis has shown that a few protein spots are changed (pI shift, appearance or disappearance of a spot) as a consequence of the application of the treatments that store or recall a meristem-production signal in flax seedlings. A SIMS investigation has revealed that the pI shift of one of these spots is probably due to protein phosphorylation. Modifications of the proteome have also been observed in Arabidopsis seedlings subjected to stimuli such as cold shock or radiation from a GSM telephone.

METHODOLOGICAL DEVELOPMENTS FOR APPLICATION TO THE STUDY OF PHYSIOLOGICAL BORON AND TO BORON NEUTRON CAPTURE THERAPY

The combination of immunogold labelling with electron microscopy or the direct detection of boron by electron energy loss spectrometry have the best lateral resolution for the imaging of boron or boron binding sites in tissues at the sub-cellular level. However these methods do not discriminate the boron isotopes. A number of physical methods make it possible to combine analytical imaging with isotopic labelling for boron studies in biological material. Secondary ion mass spectrometry has the potential to isotopically localise virtually any element with a resolution of ∼250nm with conventional instruments and 20–50nm with prototype instruments or with the NanoSIMS50; although SIMS has a relatively poor sensitivity for boron detection in biological matrices, boron imaging in plant samples is possible. Laser microprobe mass analysis also has the potential to detect boron isotopes with a lateral resolution of 3 to 5 μm and a detection limit of a few tens of μg/g with the conventional instruments and of the order of 1ng/g with the new LARIMP system; although mass resolution of LMMS is in general not very good, the risk of interference by other ions at the level of boron masses is limited. Neutron capture radiography is probably the easiest technique for boron imaging and boron isotopic labelling studies in tissues and sometimes at the sub-cellular level, although it detects only 10B isotopes. Nuclear reactions with charged particles (nuclear reaction analysis) have the potential to detect both isotopes of boron and carry out absolute boron concentration measurements with minimal matrix effects, limited risk of interference by other nuclides, a lateral resolution of a few μm at the best, a detection limit better than 1 μg/g for 11B, of the order of 10 μg/g for 10B and an accuracy of 1 to 2% in the determination of 10B/11B isotopic ratios. Preventing the diffusion of possibly mobile forms of boron during the preparation of the biological specimens is still a difficult problem for most techniques. The appropriate application of those methods, or their mutual combination or combination with other methods has made it possible: i) to yield information about the boron concentrations and fluxes in sub-cellular compartments and support the view that the cellular transport of boron was mainly passive under the experimental conditions under consideration; ii) to image the distribution of boron and of boron binding sites in tissues and sometimes at the sub-cellular level; iii) to study the short-distance diffusion and the long-distance transport of boron in plants and to assess the role of the phloem in the long-distance transport in various plant species; iv) to determine the origin (seed reserves vs uptake by roots) of the boron present in different sub-cellular compartments. For boron neutron capture therapy of cancers, invasive techniques of boron detection and imaging are comparable to the techniques described above for the study of physiological boron; for clinical applications, non-invasive techniques to follow 10B-compounds in vivo are being developed, especially by targeting of such compound by 18F and the use of positron emission tomography or by direct detection of 10B by magnetic resonance imaging.

PHYSICAL METHODS FOR IN VITRO ANALYTICAL IMAGING IN THE MICROSCOPIC RANGE IN BIOLOGY, USING RADIOACTIVE OR STABLE ISOTOPES (REVIEW ARTICLE)

Physical methods make it possible to combine analytical imaging with isotopic labelling in biological studies. With radioactive isotopes, track- radioautography may be used (in parallel with conventional grain-density radioautography) for high lateral resolution, even with energetic β-rays; in macroradioautography, filmless methods (gaseous detectors, scintillation counters, and storage phosphor screen devices) have remarkable performances. Neutron capture radiography is used mainly for the detection and imaging of one stable isotope of a few elements which have no radioisotope of practical use. With nuclear microprobes, nuclear reaction analysis and scattering analysis may serve to discriminate between isotopes (including stable isotopes). Secondary ion mass spectrometry images any isotope of almost any element with very good detection limits and a resolution better (sometimes much better) than 1 μm. Preventing the diffusion of mobile substances during the preparation of the biological specimens is still a difficult problem.

Memorization of Abiotic Stimuli in Plants: A Complex Role for Calcium

Plants are sensitive to various abiotic stimuli (including electromagnetic radiations), to which they respond by modifying their development. The response is sometimes delayed, relative to the reception of the stimulus, which implies that the corresponding information is memorized. A few cases of such behavior are described, including that controlling the induction of meristems in the hypocotyl of flax seedlings. Using this model system, calcium has been shown to play a key role not only in stimulus sensing and the possible storage of that information, but also in its final expression. Modifications of genome expression, as well as posttranslational modifications (e.g., phosphorylation) of proteins, are involved in signal transduction and the possible memorization of information. SIMS methodology provides us with interesting experimental possibilities. The process of “ion condensation,” which has been practically ignored by biologists so far, may be involved in the memorization mechanism. A few cases of the application of plant sensitivity and information memorization to agronomical or research problems are described. The role of memorization mechanisms in the elaboration of an integrated response from plants to the many, varied stimuli that they receive permanently from their environment is discussed.

Pharmacological Evidence for Calcium Involvement in the Long-Term Processing of Abiotic Stimuli in Plants

Information about abiotic conditions is stored for long periods in plants and, in flax seedlings, can lead to the production of meristems. To investigate the underlying mechanism, flax seedlings were given abiotic stimuli that included a mechanical stimulus (by manipulation), one or two cold shocks, a slow cold treatment and a drought stress and, if these seedlings were then subjected to a temporary (1 to 3 days) depletion of calcium, epidermal meristems were produced in the seedling hypocotyls. This production was inhibited by the addition to the nutrient media of EGTA, ruthenium red, lanthanum or gadolinium that affect calcium availability or calcium transport. Use of these agents revealed a period of vulnerability in information processing that was less than 2 min for mechanical stimuli and over 5 min for other abiotic stimuli, consistent with information about mechanical stimuli being stored particularly fast. We propose that external calcium is needed for the transduction/storage of the information for meristem production whilst a temporary depletion of external calcium is needed for the actual production of meristems. Such roles for calcium would be consistent with a mechanism based on ion condensation on charged polymers.

The Role of Calcium in the Recall of Stored Morphogenetic Information by Plants

Flax seedlings grown in the absence of environmental stimuli, stresses and injuries do not form epidermal meristems in their hypocotyls. Such meristems do form when the stimuli are combined with a transient depletion of calcium. These stimuli include the “manipulation stimulus” resulting from transferring the seedlings from germination to growth conditions. If, after a stimulus, calcium depletion is delayed, meristem production is also delayed; in other words, the meristem-production instruction can be memorised. Memorisation includes both storage and recall of information. Here, we focus on information recall. We show that if the first transient calcium depletion is followed by a second transient depletion there is a new round of meristem production. We also show that if an excess of calcium follows calcium depletion, meristem production is blocked; but if the excess of calcium is in turn followed by another calcium depletion, again there is a new round of meristem production. The same stored information can thus be recalled repeatedly (at least twice). We describe a conceptual model that takes into account these findings.

Chemical Microscopy of Biological Samples by Dynamic Mode Secondary Ion Mass Spectrometry (SIMS)

3D chemical microscopy is one of the emerging applications of secondary ion mass spectrometry (SIMS) in biology. Tissues, cells, extracellular matrices, and polymer films can be imaged at present with a lateral resolution of 50 nm and depth resolution of 1 nm using the latest generation of CAMECA magnetic sector NanoSIMS 50 or with a lower lateral resolution (above 100 nm) using IMS 4f Cameca SIMS equipped with cold stage. Dynamic mode SIMS analysis is performed in ultrahigh vacuum and thus requires specific and careful preparation of biological samples aimed at preserving and minimizing destruction of the original structural and chemical properties of the samples. Here we describe a methodology based on the ultrafast plunge-freezing of biological tissues, preparation of the sample for SIMS analyses and transfer to the SIMS cold stage without interruption of the cold chain during the mounting procedure and subsequent SIMS analyses. Using this strategy, SIMS chemical microscopy can be performed on biological tissue in which unwanted molecular and/or structural reorganization, loss of constituents and chemical modifications are minimized and in which structures are therefore optimally preserved.

SIMS imaging of Ca and Na, and effect of the ionic interaction Ca/Na on wall maturation of phloem fibres and xylem cells of hypocotyls of beech seedlings

Abstract Using SIMS microscopy an unexpected ionic interaction between calcium and sodium has been characterised within the walls of the xylem cells and of the phloem fibres of hypocotyls of beech seedlings: whereas a decrease of the Na signal in the cell walls was always paralleled by an increase of the calcium signal at the same site, unexpectedly an increase of the normalised Na signal in the cell walls of the Naenriched seedlings was paralleled by an increase of the Ca signal. We have also shown that this interaction was involved in the differentiation of the secondary walls.