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.

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.

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 two min for mechanical stimuli and over five 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.

Dynamic-SIMS imaging and quantification of inorganic ions in frozen-hydrated plant samples.

We present here SIMS images of the distribution of inorganic cations (Na, K, Mg and Ca) in frozen-hydrated samples of three plant species, ivy, camomile, and flax. The samples were cryofixed using fast plunge-freezing. Stigmatic images were obtained, at 100 K, under dynamic SIMS conditions by fast atom bombarding (FAB). Even though the images obtained with the frozen-hydrated plant samples are still not of upper quality, they show that the method used to prepare these samples preserves existing ionic gradients between the outer and the inner part of the cells, between adjacent cells, including cells with the same type of differentiation, and between tissues. We also describe the quantification of the relative proportions of the ions in the vacuoles of flax. The reasonable accuracy achieved for quantification of the vacuole ion ratios permitted to show (i) that radial gradients of ion ratios in hypocotyls change when the plant is becoming older and (ii) that large differences may exist between adjacent cortical cells of the same type. The role of these substantial differences in vacuole ion balance ratios is a largely unexplored issue in plant physiology.

Plant sensitivity to low intensity 105 GHz electromagnetic radiation.

Exposing seedlings of the flax, Linum usitatissimum L., to a variety of weak environmental stresses followed by a 2 day calcium deprivation, triggers the common response of production of epidermal meristems (actively dividing groups of cells) in the hypocotyl, which is the part of the stem between the root and the cotyledons (the pre-existing leaves in the embryo). This production reaches a plateau of 10-20 meristems after a month in the case of mechanical stimulation and cold shock. Recently, we have shown that radiation from a global system for mobile communication (GSM) telephone also triggers production of meristems with a plateau of around six meristems. Here, we show that a single 2 h exposure to radiation emitted at 105 GHz at non-thermal levels by a Gunn oscillator induces meristem production with kinetics similar to that induced by weak environmental stimuli and radiation from GSM telephone.

Two-dimensional electrophoresis investigation of short-term response of flax seedlings to a cold shock.

The flax, Linum usitatissimum L., is particularly suitable for studying the transduction and long-term signal storage of environmental signals. To investigate the underlying molecular mechanisms, we have focused on the initial changes in the proteome since these offer the possibility of reflecting the plant's history of exposure to stress. In principle, this 'proteome signature' might be revealed by two-dimensional electrophoresis (2-DE). We have therefore determined the potential of 2-DE to study the kinetics of changes to the proteome of flax induced by a 1 min cold shock. Protein identification is difficult with flax because of the lack of knowledge of gene sequences. Nevertheless, 2-DE analysis can be informative providing the significance of changes can be evaluated. We have developed a stringent threshold method to determine the significance of changes in gels obtained with proteins extracted from hypocotyls at different times after cold shock. This allowed us to reliably detect and characterize the kinetics of a set of seven spots that responded to cold shock and that constitute candidates for a proteome signature of long-term signal storage.

Appraisal of SIMS applicability to boron studies in plants.

In the search for a new methodological approach applicable to the determination of the still poorly known primary role of boron in plant physiology, we have undertaken to appraise the potential of the SIMS method for the analytical imaging of the boron isotopes, (10)B and (11)B, at physiological concentrations in plants. With our own, CAMECA IMS4F SIMS ion analyser, and using O(2)(+) as primary ions for the detection of B(+) (plus (12)C(+) and (40)Ca(+)) secondary ions, we have been able to map quantitatively the two boron isotopes in control and boron-enriched plants, to evaluate boron concentrations at the level of individual cells and to determine boron isotopic ratios. This provides the opportunity to carry out the simultaneous labeling and imaging of boron, using enrichment with the stable isotopes, (10)B and (11)B. The method has also the potential for the simultaneous, quantitative detection of the boron isotopes and of the borate-binding sites in plant cells.