New insight into silica deposition in horsetail (Equisetum arvense)
© Law and Exley; licensee BioMed Central Ltd. 2011
Received: 15 April 2011
Accepted: 29 July 2011
Published: 29 July 2011
The horsetails (Equisetum sp) are known biosilicifiers though the mechanism underlying silica deposition in these plants remains largely unknown. Tissue extracts from horsetails grown hydroponically and also collected from the wild were acid-digested in a microwave oven and their silica 'skeletons' visualised using the fluor, PDMPO, and fluorescence microscopy.
Silica deposits were observed in all plant regions from the rhizome through to the stem, leaf and spores. Numerous structures were silicified including cell walls, cell plates, plasmodesmata, and guard cells and stomata at varying stages of differentiation. All of the major sites of silica deposition in horsetail mimicked sites and structures where the hemicellulose, callose is known to be found and these serendipitous observations of the coincidence of silica and callose raised the possibility that callose might be templating silica deposition in horsetail. Hydroponic culture of horsetail in the absence of silicic acid resulted in normal healthy plants which, following acid digestion, showed no deposition of silica anywhere in their tissues. To test the hypothesis that callose might be templating silica deposition in horsetail commercially available callose was mixed with undersaturated and saturated solutions of silicic acid and the formation of silica was demonstrated by fluorimetry and fluorescence microscopy.
The initiation of silica formation by callose is the first example whereby any biomolecule has been shown to induce, as compared to catalyse, the formation of silica in an undersaturated solution of silicic acid. This novel discovery allowed us to speculate that callose and its associated biochemical machinery could be a missing link in our understanding of biosilicification.
KeywordsBiosilicification biogenic silica silicic acid horsetails callose PDMPO fluorescence acid digestion.
Silicon is the second most abundant element of the Earth's crust after oxygen and, perhaps surprisingly, its essentiality in biota remains equivocal . The difficulty in ascribing true biochemical essentiality to silicon probably emanates from a lack of demonstration of any silicon-requiring biochemistry and specifically Si-C, Si-O-C, Si-N, et c. bonds in any form of extant life . However, in spite of such limitations the essentiality of silicon in plants remains the subject of rigorous debate [3, 4] as do elaborations of the underlying mechanisms. Biosilicification was recently defined as 'the movement of silicic acid from environments in which its concentration does not exceed its solubility (< 2 mM) to intracellular or systemic compartments in which it is accumulated for subsequent deposition as amorphous hydrated silica'  and a number of plants are known biosilicifiers . One of the best known of these are the horsetails, Equisetum sp., and silica deposition in the tissues of these plants has been studied extensively [6–12], perhaps the seminal work in the field being carried out by Perry and Fraser . In this work scanning and transmission electron microscopy was used to illuminate the elaborate and detailed micromorphology and ultrastructure of silicas extracted from different regions of the horsetail, Equisetum arvense. The images of silicified stomata and other silica sculptures are truly breathtaking and the level of organisation of silica in the tissues prompted the authors to speculate that 'the silica acts as an in vivo stain, faithfully reproducing the organic matrix skeleton at the microscopic and macroscopic levels without staining'. Perry and Lu (1992) suggested that the organic matrix in question might be made from polymers of carbohydrates, for example, cellulose , and this suggestion was reinforced recently by Fry and colleagues who speculated that the hemicellulose, callose, in horsetail cell walls might be a potential site of silica deposition . Many different biomolecules, often having originally been extracted from biogenic silica, have been shown to accelerate or catalyse silica deposition in saturated solutions of silicic acid . However, biosilicifiers, such as horsetails, harvest silicic acid from solutions which are far from saturation and deposit it as amorphous hydrated silica and it is the elucidation of this mechanism which remains the 'Holy Grail' of biological silicification research .
Herein we have taken inspiration from the work of Perry and Fraser  on horsetail and we have used fluorescence microscopy to investigate biosilicification in horsetail and to identify the organic matrix involved in templating silica deposition in this plant.
PDMPO as a fluorescent marker of biosilicification
PDMPO as a fluorescent indicator of silica formation in vitro
The fluor PDMPO has been used to identify silica deposition in horsetail and to provide new insight into silicification in this plant. It was observed that silica deposition in horsetail exactly mirrored the known deposition of callose in the related fern and other plants. Callose was shown to induce the formation and precipitation of silica in undersaturated solutions of silicic acid. This was the first time that this had been demonstrated for any biomolecule and it suggested that callose and perhaps other similar carbohydrates might be key molecules in biological silicification.
Hydroponic culture of horsetail
Horsetail (Equisetum arvense) rhizomes were collected locally, washed in ultrapure water (conductivity < 0.067 μS/cm) and subjected to hydroponic culture in 1/6th MS medium in the presence (2 mM) or absence of added silicic acid. The latter media included an additional 8 mM Na+ to account for Si addition as Na4SiO4. After 10-12 weeks of a 14 h light/10 h dark cycle at 25°C healthy horsetail plants had grown under both sets of conditions.
Digestion of horsetail materials
Horsetail plants, either collected locally or grown hydroponically, were washed in ultrapure water, allowed to air-dry, cut into discrete 1 cm sections of rhizome/root, basal stem, distal stem, nodal regions and leaves and ca 0.5 g of each placed in acid-washed 20 mL PFA teflon© vessels. The samples were then digested in a 1:1 mixture of 15.8M HNO3 and 18.4M H2SO4 using a Mars Xpress microwave oven (CEM Microwave Technology Ltd.). The acid digests were clear and, upon dilution with 8 mL of ultrapure water, were filtered and the residues washed several times with further volumes of ultrapure water. Filters were then placed in plastic Petri dishes in an incubator at 37°C to achieve dryness over several days. Dry residues off each filter were then collected into Bijoux tubes and stored in a dry, sealed, perspex cabinet.
PDMPO labelling of horsetail silica
Silica residues collected from filters were suspended in 20 mM PIPES at pH 7 and containing 0.125 μM 2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl) -methoxy)phenyl)oxazole (PDMPO; LysoSensor Yellow/Blue DND-160, 1 mM in DMSO). This intracellular pH probe  has been shown to be bound by silica (but not silicic acid) and to emit 'green' fluorescence upon excitation at 338 nm [35–38]. Suspensions were left for 24 h to allow the reaction between silica surfaces and PDMPO to achieve completion after which 50 μL samples were transferred to a cavity slide and viewed using an Olympus BX50 fitted with a BXFLA fluorescent attachment using a U-MWU filter cube (Ex: 333-385 nm; Em: 400-700 nm). A ColourView III digital camera (OSIS FireWire Camera 3.0 digitizer) was used to capture images in conjunction with CELL* Imaging software (Olympus Cell* family, Olympus Soft Imaging solutions GmbH 3.0).
In vitro preparations of callose and silicic acid
Callose (β-D Glucan, Barley, Sigma, UK) was dissolved at 5% w/v in 20 mM PIPES buffer solutions at pH 7 and containing 0, 1, 2, 4 and 7 mM Si(OH)4 by warming each preparation in a water bath at 100°C for 60 seconds. Upon cooling to room temperature PDMPO was added to a concentration of 0.125 μM. Equivalent control solutions to which no callose had been added were treated in an identical manner. All solutions were then incubated at room temperature in the dark for 5 days before being examined by fluorescence microscopy, see above, or their emission spectra were determined by fluorimetry (Perkin-Elmer LS50B; Ex; 338 nm; Em: 400-650 nm) as previously described .
CL was in receipt of a NERC studentship.
- Exley C: Silicon in life: a bioinorganic solution to bioorganic essentiality. J Inorg Biochem. 1998, 69: 139-144. 10.1016/S0162-0134(97)10010-1.View Article
- Exley C: Darwin, natural selection and the biological essentiality of aluminium and silicon. Trends Biochem Sci. 2009, 34: 589-593. 10.1016/j.tibs.2009.07.006.PubMedView Article
- Epstein E: The anomaly of silicon in plant biology. Proc Natl Acad Sci USA. 1994, 91: 11-17. 10.1073/pnas.91.1.11.PubMedPubMed CentralView Article
- Currie HA, Perry CC: Silica in plants: biological, biochemical and chemical studies. Ann Bot. 2007, 100: 1383-1389. 10.1093/aob/mcm247.PubMedPubMed CentralView Article
- Exley C: Silicon in life: whither biological silicification?. Biosilica in Evolution, Morphogenesis and Nano-biotechnology. Edited by: Muller WEG, Grachev MA. 2009, Springer, 173-184.View Article
- Page CN: An assessment of inter-specific relationships in Equisetum subgenus Equisetum. New Phytol. 1972, 71: 355-369. 10.1111/j.1469-8137.1972.tb04082.x.View Article
- Kaufman PB, LaCroix JD, Dayanandan P, Allard LF, Rosen JJ, Bigelow WC: Silicification of developing internodes in the perennial scouring rush (Equisetum hyemale var. affine). Developmental Biol. 1973, 31: 124-135. 10.1016/0012-1606(73)90324-2.View Article
- Laroche J, Guervin C, Le Coq C, Robert D: Activités pétrogénétiques chez Equisetum arvense L. (Ptéridophytes). Bulletin de la Société Botanique de France. 1992, 139: 47-55.View Article
- Holzhüter G, Narayanan K, Gerber T: Structure of silica in Equisetum arvense. Analyt Bioanalyt Chem. 2003, 376: 512-517. 10.1007/s00216-003-1905-2.View Article
- Sapei L, Nöske R, Strauch P, Paris O: Isolation of mesoporous biogenic silica from the perennial plant Equisetum hyemale. Chem Mat. 2008, 20: 2020-2025. 10.1021/cm702991f.View Article
- Gierlinger N, Sapei L, Paris O: Insights into the chemical composition of Equisetum hyemale by high resolution Raman imaging. Planta. 2008, 227: 969-980. 10.1007/s00425-007-0671-3.PubMedPubMed CentralView Article
- Currie HA, Perry CC: Chemical evidence for intrinsic 'Si' within Equisetum cell walls. Phytochemistry. 2009, 70: 2089-2095. 10.1016/j.phytochem.2009.07.039.PubMedView Article
- Perry CC, Fraser MA: Silica deposition and ultrastructure in the cell wall of Equisetum arvense: the importance of cell wall structures and flow control in biosilicification?. Phil Trans Roy Soc London B. 1991, 334: 149-157. 10.1098/rstb.1991.0104.View Article
- Perry CC, Lu Y: Preparation of silicas from silicon complexes: Role of cellulose in polymerisation and aggregation control. J Chem Soc Faraday Trans. 1992, 88: 2915-2921. 10.1039/ft9928802915.View Article
- Fry SC, Nesselrode BHWA, Miller JG, Mewburn BR: Mixed linkage (1→3,1→4)-β-glucan is a major hemicellulose of Equisetum (horsetail) cell walls. New Phytol. 2008, 179: 104-115. 10.1111/j.1469-8137.2008.02435.x.PubMedView Article
- Perry CC: An overview of silica in biology: Its chemistry and recenttechnological advances. In Biosilica in Evolution, Morphogenesis and Nanobiotechnology.Edited by: Muller WEG, Grachev MA. Springer; 2009:295-313.View Article
- Chen CH, Lewin J: Silicon as a nutrient element for Equisetum arvense. Can J Bot. 1969, 47: 125-131. 10.1139/b69-016.View Article
- Fauteux F, Chain F, Belzile F, Menzies JG, Belanger RR: The protective role of silicon in the Arabidopsis-powdery mildew pathosystem. Proc Natl Acad Sci USA. 2006, 103: 17554-17559. 10.1073/pnas.0606330103.PubMedPubMed CentralView Article
- Apostolakos P, Livanos P, Galatis B: Microtubule involvement in the deposition of radial fibrillar callose arrays in stomata of the fern Asplenium nidus L. Cell Motility Cytoskeleton. 2009, 66: 342-349. 10.1002/cm.20366.View Article
- Apostolakos P, Livanos P, Nikolakopoulou TL, Galatis B: The role of callose in guard-cell wall differentiation and stomatal pore formation in the fern Asplenium nidus. Ann Bot. 2009, 104: 1373-1387. 10.1093/aob/mcp255.PubMedPubMed CentralView Article
- Apostolakos P, Livanos P, Nikolakopoulou TL, Galatis B: Callose implication in stomatal opening and closure in the fern Asplenium nidus. New Phytol. 2010, 186: 623-635. 10.1111/j.1469-8137.2010.03206.x.PubMedView Article
- Scherp P, Grotha R, Kutschera U: Occurrence and phylogenetic significance of cytokinesis-related callose in green alga, bryophytes, ferns and seed plants. Plant Cell Rep. 2001, 20: 143-149. 10.1007/s002990000301.View Article
- Thiele K, Wanner G, Kindzierski V, Jürgens G, Mayer U, Pachl F, Assaad FF: The timely deposition of callose is essential for cytokinesis in Aribodopsis. The Plant J. 2009, 58: 13-26. 10.1111/j.1365-313X.2008.03760.x.PubMedView Article
- Chen XY, Kim JY: Callose synthesis in higher plants. Plant Signall Behav. 2009, 4: 489-492. 10.4161/psb.4.6.8359.View Article
- Fry SC, Mohler KE, Nesselrode BHWA, Franková L: Mixed linkage β-glucan: xyloglucan endotransgluocosylase, a novel wall remodelling enzyme from Equisetum (horsetail) and charophytic algae. The Plant J. 2008, 55: 240-252. 10.1111/j.1365-313X.2008.03504.x.PubMedView Article
- Sørensen I, Pettolino FA, Wilson SM, Doblin MS, Johansen B, Bacic A, Willats WGT: Mixed linkage (1→3, 1→4)- β-D-glucan is not unique to the Poalesm and is an abundant component of Equisetum arvense cell walls. The Plant J. 2008, 54: 510-521. 10.1111/j.1365-313X.2008.03453.x.PubMedView Article
- Xu XM, Jackson D: Lights at the end of the tunnel: new views of plasmodesmal structure and function. Current Opin Plant Biol. 2010, 13: 684-692. 10.1016/j.pbi.2010.09.003.View Article
- Zavaliev R, Ueki S, Epel BL, Citovsky V: Biology of callose (β-1,3-glucan) turnover at plasmodesmata. Protoplasma. 2011, 248: 117-130. 10.1007/s00709-010-0247-0.PubMedView Article
- Nishikawa SI, Zinkl GM, Swanson RJ, Maruyama D, Preuss D: Callose (beta-1,3-glucan) is essential for Aribodopsis pollen wall patterning, but not tube growth. BMC Plant Biol. 2005, 5: art 22.View Article
- Majewska-Sawka A, Münster A, Rodríguez-García MI: Guard cell wall: immunocytochemical detection of polysaccahride components. Journal of Experimental Botany. 2002, 53: 1067-1079. 10.1093/jexbot/53.371.1067.PubMedView Article
- Thompson MV, Wolniak SM: A plasma membrane-anchored fluorescent protein fusion illuminates sieve element plasma membranes in Aribodopsis and tobacco. Plant Physiol. 2008, 146: 1599-1610. 10.1104/pp.107.113274.PubMedPubMed CentralView Article
- Cui SW, Wang Q: Cell wall polysaccharides in cereals: chemical structures and functional properties. Struct Chem. 2009, 20: 291-297. 10.1007/s11224-009-9441-0.View Article
- Størseth TR, Kirkvold S, Skjermo J, Reitan KI: A branched β-D-(1→3, 1→6)-glucan from the marine diatom Chaetoceros dibilis (Bacillariophyceae) charcaterised by NMR. Carbohydrate Res. 2006, 341: 2108-2114. 10.1016/j.carres.2006.05.005.View Article
- Diwu Z, Chen CS, Zhang C, Klaubert DH, Haugland RP: A novel acidotropic pH indicator and its potential application in labeling acidic organelles of live cells. Chem and Biol. 1999, 6: 411-418. 10.1016/S1074-5521(99)80059-3.View Article
- Shimizu K, Del Amo Y, Brzezinski MA, Stucky GD, Morse DE: A novel fluorescent silica tracer for biological silicification studies. Chem Biol. 2001, 8: 1051-1060. 10.1016/S1074-5521(01)00072-2.PubMedView Article
- Leblanc K, Hutchins DA: New applications of a biogenic silica deposition fluorophore in the study of eceanic diatoms. Limnol Oceanography::Methods. 2005, 3: 462-476.View Article
- Hazelaar S, van der Strate HJ, Gieskes WWC, Vrieling EG: Monitoring rapid valve formation in the pennate diatom Navicula salinarum (Bacillariophyceae). J Phycol. 2005, 41: 354-358. 10.1111/j.1529-8817.2005.04131.x.View Article
- Ogane K, Tuji A, Suzuki N, Kurihara T, Matsuoka A: First application of PDMPO to examine silicification in polycystine Radiolaria. Plankton Benthos Res. 2009, 4: 89-94. 10.3800/pbr.4.89.View Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.