Study of the mechanism of diatom cell division by means of 29Si isotope tracing J.- N. Audinota*, C. Guignardb, H.- N. Migeona and L. Hoffmannb. a Laboratoire d’Analyse des Matériaux, Centre de Recherche Public – Gabriel Lippmann 41, rue du Brill, L-4422 Belvaux, Luxembourg b Cellule de Recherche en Environnement et Biotechnologies, Centre de Recherche Public – Gabriel Lippmann, 41, rue du Brill, L-4422 Belvaux, Luxembourg firstname.lastname@example.org Abstract Diatoms are delicate unicellular organisms enclosed in a silica frustule, that is made up of two valves. Multiplication of the diatoms occurs by ordinary mitotic cell division. During cell division each cell produces two daughter cells, each of them keeping one of the two valves of the mother cell and producing a new valve by absorbing the silicon present in the environment. The NanoSIMS 50 allows ion imaging to be performed on diatoms in order to determine the site of fixation of silicon. The aim of this study was to observe and compare the mechanism of the construction of the new valve after cell division. To this end, different types of diatoms have been transferred in a culture medium enriched with 29Si and after several days, the distribution of the different isotopes of silicon has been determined by NanoSIMS50 imaging. The construction of new valves has been observed and the isotopic ratio has been determined. Keywords: NanoSIMS, isotope tracing, diatoms, biological sample, silicon 1. Introduction The lateral resolution (around 50nm) of the NanoSIMS as well as very high transmission at high mass resolution (80% at M/∆M =5 000) render this ion microscope a tool of choice for the study of isotopic elements in biological tissues [1-3]. Diatoms are delicate unicellular organisms that have a yellow-brown chloroplast that enables them to photosynthesize. Most diatoms are photosynthetic micro-organisms, although some may live heterotrophically. Although unicellular, they may form branched or filamentous colonies, and even be embedded in a gelatinous envelope or tube. All diatoms are enclosed in a silica frustule, that is made up of two valves fitted together by a connective zone called a girdle. Multiplication of the diatoms generally occurs by ordinary mitotic cell division. During cell division each cell produces two daughter cells, each of them keeping one of the two valves of the mother cell and producing a new valve by absorbing the silicon present in the environment. The aim of this study was to understand, observe and compare the mechanism of the construction of the missing valve. To this end, different types of diatoms have been transferred in a culture medium enriched with 29Si for several days (1-5 days). The distribution of the isotopes of silicon (28Si, 29Si and 30 Si) has been determined by NanoSIMS50 imaging. 2. Experimental Preparation of the 29Si-enriched culture medium Labelled silica (96.74% atom 29Si) was purchased from Euriso-top (Saclay, France). This labelled silicium oxide (19.4 mg) was converted into potassium metasilicate (K2SiO3) by alkaline melting with 300 mg KOH in a nickel crucible, then dissolved in 80 mL DI-water (18.2 MΩ.cm-1) and neutralized with hydrochloric acid (HCl 37 % aqueous solution). The volume of the solution was adjusted to 100 mL (concentration: 3.172 mmol/L 29Si). The enriched culture mixture was prepared by adding 4 mL of this 29Si solution to 96 mL of standard diatom culture medium containing 0.134 mmol/L Si . The final 29Si/28Si ratio after enrichment was 1.08. Diatom growth Diatoms (Nitzschia palea) were obtained from the Diatom collection of the Public Research Center – Gabriel Lippmann. An aliquot (2 mL) of the diatom suspension was put in a sterile culture flask and 10 mL of labelled culture medium were added. During the incubation period, the culture flask was kept at room temperature (21°C) under constant light. Sample preparation The culture flask was shaken during 5 minutes to detach all diatoms and homogenize the culture medium, then 5 mL were transferred into a 16 cm x 1.6 cm i.d. test tube and 15 mL of hydrogen peroxide (H2O2 35 % aqueous solution) were added. The temperature of the mixture was increased to 90°C and maintained during 4 hours to ensure a complete oxidation of the organic content. After 4 hours of sedimentation at room temperature, the upper layer was discarded. To remove carbonate residues, 5 mL of hydrochloric acid (HCl 37 % aqueous solution) were added and allowed to react during 2 hours at room temperature. Finally, 10 mL of DI-water were added to rinse the frustules, then the upper layer was discarded after 4 hours of sedimentation. This step was reiterated 3 times to obtain a satisfactory removal of HCl, H2O2 and other salts initially contained in the culture medium. The bottom layer of the resulting solution, containing prepared frustules, was deposited with a Pasteur pipette on a copper plot heated at 60°C. After complete drying, the sample was analyzed. SIMS analysis The distribution of the isotopes of silicon (28Si, 29Si and 30Si) has been obtained in the multicollection mode with parallel detection of 16O- and 12C2- at a mass resolution of 3500 (to minimize 28 1 Si H contribution on 29Si). Maps have been acquired under standard analytical conditions, a Cs+ ion primary beam with impact energy of 16keV and a current of 0.8 pA. All images have been acquired in 256 x 256 pixels with a counting time of 20 ms by pixel. 3. Results The control diatoms grown in the standard culture medium were studied first. In this sample the isotopic images 29Si and 28Si have been recorded (figures 1a and 1b); the ratio image 29Si/28Si (figure 1c) shows an homogeneous distribution across the diatoms. The discrepancy between the “as measured ratio” of 0.07 and the natural ratio of 0.05 is attributed primarily to the different gains of the two detectors and, possibly, to some 28Si1H contribution on 29Si. Ion images of diatoms from the culture medium enriched with 29Si are shown on figures 2 and 3. Diatoms, at different moments of their life cycle, can be observed on figure 2: valves with an isotopic ratio 29Si/28Si close to 0.05 characteristic of the mother diatom valve and a diatom valve with a high concentration of 29Si (figure 2b, white arrow), characteristic of a newly formed valve. The ratio between the 29Si-image and the 28Si-image is shown in figure 2d. It can be concluded that the with 29Si enriched valve is a newly formed valve and that to construct this new valve, the silicon of the 29Si- enriched culture medium has been absorbed during the multiplication. Figure 1 : 28Si (left) and 29Si (center) distribution in control diatoms (image field 30 x 30µm2, linear (a) (b) scale). Right: ratio image 28/29 using a logarithmic scale. Valve enriched 29 Si/28Si ≈1 (a) (b) (c) (d) Figure 2: 28Si (a), 29Si (b), 30Si(c) and isotopic ratio 29Si/28Si (d) distribution in diatoms after culture in medium enriched in 29Si (image field 30 x 30µm2, logarithmic scale). (a) (b) (c) 1.5 1.4 29Si/28Si 30Si/28Si 1.3 Labelled medium 29Si/28Si=1.1 1.2 1.1 29 28 Labelled medium Si/ Si = 1 1 0.9 Isotopic ratio 0.8 0.7 0.6 0.5 28 28 Natural 29Si/Si Si=0.05 29 Natural Si/ = 0.05 0.4 Natural 30Si/28Si=0.03 30 28 Natural Si/ Si = 0.05 0.3 0.2 0.1 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 d (µm) Figure 3 : 28Si (a) and 29Si (b) maps, isotopic ratio 29Si/28Si (c) (log scale) and line-scan 29/28 (as depicted on image a) in diatoms after culture in medium enriched with 29Si. Different analyses have been realized on the same diatoms solution. In some cases, an inhomogeneous distribution in 29Si has been found in some valves (figure 3). In this analyzed area, valves of diatoms are more or less enriched in 29Si (figures 3a and 3b), as is also shown in the image of the isotopic ratio 29Si/28Si (figure 3c). This representation highlights the inhomogeneous distribution in 29 Si. Conclusion These preliminary measurements have confirmed the multiplication of diatoms by ordinary mitotic cell division. Furthermore, the lateral resolution provided by the SIMS experiments permits to record heterogeneous distributions of the silicium 29/28 ratio inside one single valve. Together with improved accuracy of the isotopic ratio measurements this will permit, in future works, to investigate in details the site of fixation of silicon (growth mechanism) up to the construction of the complete missing valve. Acknowledgements This work received the financial support of the Fonds National de Recherche de Luxembourg in the framework of project FNR/01/02/01b. F. Rimet is thanked for providing the diatom culture. References  F. Hillion, B. Daigne, F. Girard, G. Slodzian, in: Proceedings of the SIMS IX, Yokohama, 1993, p. 254.  N. Grignon, J.J. Vidmar, F. Hillion, B. Jaillard, in: Proceedings of the SIMS XII, Brussels, 1999, p. 903.  J.- L. Guerquin-Kern, T.- Di Wu, C. Quintana and A. Croisy, accepted for publication in Biochimica and Biophysica Acta (2005).  R.R.L. Guillard and C. L. Lorenzen. J. Phycol., 8 : 10-14 (1972).
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