Equisetum_01

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Abstract Silicified regions in the stem and leaf of thehorsetail Equisetum arvense were studied by scanning andtransmission electron microscopy. The silica was presentas a thin layer on the outer surface with variation in thesize of this layer depending on the part investigated. Therewas a dense arrangement of silica spheres with some den-sity fluctuations. A loose arrangement of silica particleswith variation in their size was found beneath this densearrangement suggesting the agglomeration of silica. Anelectron diffraction pattern showed the presence of amor-phous silica, with the short range order being comparableto that of silica from other chemical sources. The mediumrange order shows the presence of silica with a high inner surface. SAXS measurements correlate with the particlesize observed in TEM, and provide values for surfacefractals. A new method of plasma ashing to remove theorganics is also described.

Keywords Silica · Horsetail · Biomineralisation ·Electron microscopy · SAXS

Introduction

In nature, silica can be found in higher terrestrial plantsand a wide variety of organisms like diatoms, sponges andmolluscs [1, 2]. De Saussure (in Ref. [3]) first describedthe silicification in plants. Silica distribution in variousgrasses [4, 5, 6] has been widely studied and reported. Theshapes of phytoliths are characteristic of a given plant and

vary between different species [7, 8]. Usually silica isfound in parts of plants associated with cell wall in thesubterranean and aerial organs, those that infill the cell lu-men and as extra cellular deposits. These silica depositscan reproduce or mimic carbon-based cell structures in

three dimensions; such replacement structures are retainedlong after the death of the plant and can be used to iden-tify the precursor plant. Although silica may not be neces-sary for healthy growth of the plants, it has been found tohave some secondary effects. It has been speculated thatbiomineralised silica may play a structural role or act as amineral barrier to both invasion of pathogens and translo-cation of water and salts.

No other plant has a higher concentration of silica thanthe horsetail [9]. It is one of the oldest plants on earth andwhat remains today from tree-sized fossils are the fieldhorsetails. They were used in historical times for scouringpots and polishing pewter and were commonly called“scouring rushes”. Horsetails have found extensive appli-cation in medicine as a source of silica, as it can amountto 25% of the dry weight of the plant. Researchers [10, 11]believe that the medicinal property of horsetail is due to

its high silica content. It has good antibacterial, antisepticand astringent properties. In many plants dissolved silicaappears to be taken into the plant as an inert componentand is deposited in places where water evaporates. In anycase, the structure of silica determines the property and us-age. But the mechanism of silica accumulation in theplants [12] still remains poorly studied although variouspossible mechanisms have been proposed. Previous re-search on silica distribution in horsetails was mostly basedon scanning electron microscopy [13] and the importanceof structure–function relation and cell wall composition inhorsetail [14] has been discussed. The associated macro-molecules which might be the structure-directing agents

seem to be unknown. Biosilicification and the role of theorganic matrix in controlling the structure has been re-viewed in detail by Perry et al. [15].

In previous studies, mostly ground horsetail sampleswere taken for study which does not focus on the point of interest. In the current study, we examine sections acrossvarious significant silica structures using a number of dif-ferent methods for structural investigation. A structure cor-relation to the investigating methods has been attempted.

G. Holzhüter · K. Narayanan · T. Gerber

Structure of silica in Equisetum arvense

Anal Bioanal Chem (2003) 376: 512–517DOI 10.1007/s00216-003-1905-2

Received: 27 September 2002 / Revised: 3 March 2003 / Accepted: 11 March 2003 / Published online: 6 May 2003

ORIGINAL PAPER

G. Holzhüter (✉) · K. Narayanan · T. Gerber Department of Physics, University of Rostock,18051 Rostock, Germanye-mail: [email protected]

© Springer-Verlag 2003



Experimental

Materials and methods

Samples of shoots of Equisetum arvense were collected in autumnand spring. Shoots were separated into stems and leaves. A part of the sample was fixed with glutaraldehyde and subjected to criticalpoint drying so as to retain the cell structures without significantdeformation. A part of this fixed sample was further fixed withOsO4 for contrasting between the organic part and silica and thenembedded in epoxy resin. Ultrathin sections of both longitudinaland axial parts were made with a diamond knife on an ultramicro-tome and placed on copper grids. These sections were viewed in aLEO 912 TEM operated at 100keV and electron diffraction wasmade on the same instrument. TEM images and the diffraction pat-tern were recorded on image plates. Gold was used as the standardfor calibration. From the diffraction pattern of the samples andgold standard, radial intensity scans were made. The scattering in-tensities were normalised and then Fourier transformed to differ-ence atomic radial distribution functions [16, 17, 18]. Sampleswere mounted on aluminium stubs and coated with gold for analy-

sis by scanning electron microscopy. For elemental mapping, thesamples were taken as such without gold sputtering. In order to re-move the organics for improved examination of the silica layer,natural samples (without any fixation) were plasma ashed in anoxygen atmosphere in a K1050 Plasma asher. The samples wereplasma ashed for 4.5 h with a power of 150 W. Plasma ashed andnatural samples were lightly ground with a pestle and mortar andthe powdered sample was subjected to SAXS studies. SAXS mea-surements were performed on a Kratky system using Ni-filteredCu Kα radiation (sealed tube, 2kW X-ray generator); data werecollected and analysed by a previously described protocol [19].

Results and discussion

Localisation of silica

Elemental mapping of silicon was done on samples thathad been subjected to critical point drying to identify po-sitions where silica was concentrated. Studies on severalparts of the stem and leaves gave impressive results. Fig-ure 1a shows the SEM of cross section of the stem from

Equisetum arvense. Figure 1b shows the silicon distribu-tion in the regions of Fig. 1a. In general, the relative posi-tioning of silica was as a thin layer on the surface. Andthis thin layer was prominent, irrespective of the part of

the plant examined. This can be because, once the silica isconcentrated and converted to colloidal forms, it cannotenter the cell membranes and so remains concentrated assuch.

Ultra structure studies by TEM

TEM examination showed that the silica surface layer varied in thickness depending on the part of the plant, theleaf or the stem. To describe the silica layer more thor-oughly, we divided the layer into three parts – an outer-most thin layer, a dense middle region and an inner re-gion.

In general, the silica layer was found to vary in thick-ness between 3µm and 7µm in the stem and from about200nm to 1µm in the leaf (Figs.2 and 3). The outermostthin layer was found to consist of particles which resem-ble spheres without pores. The size of these spheres var-ied between 25nm and 40nm in diameter. In the middleregion, arrangement of the silica was found to be morecompact, but there were some density fluctuations in thedistribution.

When the middle layer is viewed under a higher mag-nification, new structures with some holes and channels in

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Fig. 1 (a) Cross section through thestem of equisetum arvense; scale bar 200µm. (b) Silicon distribution inFig. 1a

Fig.2 Fully mineralised outermost cell wall in the cross section of stem; scale bar 2 µm



between are visible (Fig.4). The holes seem to be thecross section of fibres. These structures are caused by thenetwork of organics. The preferred orientation of the sameis along the radial and parallel axis of the plant. This indi-cates that the organic part associated with the silica mightbe the template directing the localisation of silica particles.Earlier experiments on silica deposition [20, 21] have shownthat it is a permeation and void filling process rather thanreplacement of the cell walls, wherein the organic part

acts as template for the deposition. A mechanism for thenucleation of amorphous silica on plant materials [20] in-volving hydrogen bonding between the hydroxyl groupsin the silicic acid and cellulose, lignin within the organictissue has also been proposed. The silica with the associ-ated organic molecules could account for the mechanicalstrength of the plant and the future work should addressthe analysis of this organic part.

Towards the inner region a transition was found fromthe dense structure to a more loose arrangement of sepa-rated particles (Fig.5). These particles were principally of two different sizes, larger particles of about 60nm in di-

ameter and smaller particles with a diameter of 8nm. Itcould be hypothesised that as the plant grows and there isincreasing water movement, these separate particles could

aggregate more towards the outer region. As they ag-glomerate, these particles could influence the density fluc-tuation in the middle part.

Structure–function relation

The structure of silica in various parts of the horsetail, aspresented above, appears to correlate with the function of the corresponding part of the plant. The outer layer is theone which is more prone to structural damage and utilisedin the water and other exchange activities of the plant.Hence, the silica remains as a thin layer with not a very

compact arrangement. It could be that the smaller parti-cles have been leached away, leaving behind the larger particles which remain as the thin surface layer. In case of the middle layer, the compact dense arrangement could aidin the stiffness of the plant and thereby prevent the attackfrom predators as well. The inner region with the smallloose silica particles could be the region where the dis-

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Fig. 3 Fully mineralised surface region of a leaf cross section;scale bar 200 nm

Fig. 4 Dense silica with pores or channels, probably caused by theorganic matrix; scale bar 30 nm

Fig.5 The inner part of the mineralised layer consisting of 60nmspheres and 8 nm silica particles; scale bar 100nm

Fig.6 Typical amorphous diffraction pattern of silica made fromthe denser structures (Fig.4)



solved silica is taken up from the soil with other elements,is getting polymerised to form small particles initially.These particles could later agglomerate and form granular spheres and as water flows towards the ends, they get con-centrated to form a dense arrangement as in the middleand outer layers.

Electron diffraction studies

The electron diffraction pattern in Fig. 6 shows the silicato be principally of amorphous form. On further analysisof the scattering intensities, we have deduced the reducedintensity function s*i(s) and show it in Fig.7. From this,the radial distribution function was determined and the in-teratomic distances estimated (Fig. 8). The first four inter-atomic distances in our sample are similar to silica struc-tures from sources like vapour deposited silica, silica geland silica glass [22], with a comparatively higher orderingthan in vapour deposited silica but less than in silica glass.The medium-range order can be described with the first peak

position in the reduced intensity function which is charac-

teristic for the amount of inner surfaces. Gerber et al. [23]have found values between 15nm –1 in case of silica glassup to 16.5nm –1 for silica gel grown at a pH value of 2.2.In the present study, the value of 16.5 nm –1 could corre-spond to a silica structure with high inner surfaces. There-fore we conclude that the silica with high inner surface inhorsetail is more similar to the ones in gel and this me-dium range order is not influenced by organic templates.

SAXS studies

SAXS measurements were done on leaf and stem parts of Equisetum arvense. This is an integral method of structureinvestigation and results from this method can confirm re-sults from TEM measurements in the range of up to 30nm.The logarithmic intensity versus q plots of these naturalsamples showed almost the same structure. In case of theplasma ashed sample, the intensity was higher by morethan 10 times and the similarity in curves proved that thewhole structure was not altered during the ashing process.

As the curves were similar, a single curve is presented

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Fig. 8 Radial distribution function of silica in horse tail

Fig.9 Double logarithmic plot of intensity of plasma ashed leaf of Equisetum arvense

Fig. 10 Plot of chord distribution function showing particles of8 nm in diameter

Fig. 7 Reduced intensity function calculated from the diffractionpattern in Fig.6



(Fig. 9) which is representative of all the samples. In thedouble logarithmic plot, the initial part of the curve is lin-ear followed by a small shoulder. This Guinier region cor-responds with particle sizes of nearly 8nm in diameter.

The chord distribution function calculation (Fig.10) yieldsparticle sizes of nearly 8 nm in diameter. On curve fitting,under the collimation conditions, the slope of the linear region obtained is 3.2. This value in the double logarith-mic plot can be correlated to the surface fractal with afractal dimension (Df ) using the formula slope=6–Df Thefractal dimension value obtained is about 2.8. We ob-served particles of this diameter in TEM investigation aswell but such particles seemed to have a loose arrange-ment. These two investigating methods can be correlatedin the way that the particles are connected at the surfacethereby having a fractal dimension value of nearly 2.8 anda loose packing as well. But specific studies with SAXS

cannot be made on a particular section of the leaf or stem,which is possible by TEM.

Plasma ashing

The silica deposits in plant cells cannot be easily studiedunder the microscope without special treatment of theplant material, either to destroy the organic matter or dis-tinguish the silica from cell wall. Various methods havealready reported the removal of organics; however, it isapparent that none of the methods were able to remove theorganics completely, and harsher treatments led to a dam-

age of silica structure. The surface of horsetail is charac-terised by a well-defined micro morphology and the vari-ous surface structures have been described in detail by Perryet al. [14]. A low-temperature plasma method [24, 25] of obtaining ash to avoid thermal effects had been reportedearlier, but has not been widely used. The current sam-ples, without any fixation, were plasma-ashed in an oxy-gen atmosphere. The highest power of 150 W for nearly4.5 h led to the removal of almost all the organics. An EDXspectrum of the plasma-ashed sample (Fig.11) substanti-ates the same. As shown by the SEM photo in Fig.12, thismethod doesnot lead to significant structural damage and

the surface morphology of the plasma-ashed sample issimilar to that of the untreated sample. This shows that thesilica mimics the underlying cell structure thereby pro-tecting the plant from predators and offering stability to

the plant as well.

Conclusions

From the elemental mapping and EDX, we can concludethat the silica is concentrated on the surface as a thinlayer. Ultra structure studies with TEM reveal the thick-ness of the surface layer to vary from a few nanometers toabout 7µm. The silica layer in the outer part was moredense due to closer packed particles. The middle layer also has a dense silica structure with some holes or chan-

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Fig. 11 EDX spectra of plas-ma ashed sample substantiatingthe removal of organics

Fig. 12 Undamaged stomata on the surface of a plasma ashedsample; scale bar 20µm



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nels in between and probably the organic part fills thesechannels. Hence this surface layer could be described as acompound structure of silica interwoven with the organ-ics. Towards the inner part, a loose arrangement of silicaparticles is found and this could be the region where thesilica accumulates. Electron diffraction shows an amor-phous silica. The RDF is typical for connected Si–O tetra-hedrons forming a network as in a silica xerogel. The re-

sulting network has high amount of inner surfaces as in adried gel produced at pH2.2. The organic part has no ef-fect on silica structure in the atomic range. From the in-tensity plot of SAXS measurements, the presence of sur-face fractal with a particle size of about 8 nm is deduced.These particles are connected at the surface in a random wayand also correspond to the particle size deduced in TEMinvestigations. Plasma ashing is one of the best methodsto remove the organics without significant surface destruc-tion and this silica layer at the surface is dense offeringstrength and protection to the plant.

Acknowledgement The authors would like to thank the DFG for funding the project 12001361 under the head Schwerpunktspro-

gramm (SPP) 1117: “Prinzipien der Biomineralisation”.

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