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BlackwellScienceLtdIntracellular calcium oxalate crystal structure inDracaena sanderiana

Svoboda V. Pennisi

1

, Dennis B. McConnell

2

, Laurie B. Gower

3

, Michael E. Kane

2

and T. Lucansky

3

1University of Georgia, Horticulture Department, Coastal Plains Experimental Station, Tifton, GA 31793, USA; 2University of Florida, Environmental

Horticulture Department, 1519 Fifield Hall, Gainesville, FL 32611– 0670, USA; 3University of Florida, Materials Science and Engineering Department, 210

Rhines Hall, Gainesville, FL 32611– 6400; 3University of Florida, Botany Department, 3191 McCarty Hall, Gainesville, FL 32611–6400, USA

Summary

• The chemistry, crystallography and ultrastructure of intracellular calcium oxalatedeposits in the angiosperm, Dracaena sanderiana are reported here.• Crystalline deposits extracted from mature leaves and leaf primordia ofD. sanderiana

were studied by scanning electron microscopy and X-ray powder diffractometry

techniques, and compared with X-ray standards for calcium monohydrate and calciumoxalate dihydrate.• Intracellular calcium oxalate deposits were of two types; calcium oxalate mono-hydrate raphides or solitary calcium oxalate dihydrate crystals. Raphide-containingcells exhibited lamellate sheaths around the chamber walls, mucilage-like materialssurrounding the developing crystal chambers, and paracrystalline bodies with closelyspaced subunits within the chambers. The intracellular calcium oxalate dihydratecrystals usually displayed typical tetragonal-dipyramidal morphology, but develop-ment of some unusual crystal faces occasionally occurred.• Two intracellular hydrate forms of calcium oxalate (monohydrate and dihydrate)exist in D. sanderiana. The elaboration of crystal vacuoles derived from rough endo-plasmic reticulum and modified crystals with energetically unfavourable facessuggest that precipitation of calcium oxalate dihydrate in D. sanderiana cells mightbe biologically controlled.

Key words: Plant crystals, biomineralization, calcium oxalate, raphides, crystalidioblast.

© New Phytologist (2001) 150: 111–120

Author for correspondence:Dennis B. McConnell

Tel: +1 352 392 7932

Fax: +1 352 392 3870

Email: [email protected]

Received: 27 April 2000 Accepted: 7 November 2000

Florida Agricultural Experiment Station journal series no. R-07536

Introduction

The most common calcium oxalate (CO) hydrates found inplants are calcium oxalate monohydrate (COM) and calciumoxalate dihydrate (COD). Angiosperms typically deposit COcrystals inside cell vacuoles of highly specialized cells. Thecrystal-containing cell is usually conspicuously larger thansurrounding cells and is termed crystal idioblast . Horner &

Wagner (1995) proposed two general systems based on thepresence or absence of membranes and associated subcellularstructures. System I was exemplified by druses in Capsicum and Vitis , raphides in Psychotria , and crystal sand in Beta .System I crystal idioblasts presented cytoplasmic spherosomes,vacuolar organic paracrystalline bodies, membrane complexes,

plasmalemmasomes, and crystal chambers. The vacuolarparacrystalline bodies exhibited subunits with large periodicity and were linked to a membrane network, which formed thecrystal chambers. System I crystal idioblasts were observedonly in dicotyledonous species. System II was exemplified by the monocotyledonous raphide idioblasts in Typha , Vanilla and Yucca . System II lacked vacuolar membrane complexes,and paracrystalline bodies displayed closely spaced subunits.Mucilage-like material was present around developing crystalchambers and lamellate sheaths were observed around cham-ber walls (Wattendorff, 1976; Horner & Wagner, 1995).

In reports where conclusive X-ray diffraction analysis wasused, the crystalline matter in plants was shown to be COM.Reported occurrences of COD crystals are scarce, and conclusive



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reports of their chemical identity are even scarcer. In mostinstances, crystal morphology has been the only definitivefeature used in analysis. Confirmed reports on intracellularCOD in plants determined by X-ray diffraction include:Capsicum annuum (Solanaceae), solitary prisms (Wagner, 1983)and druses (Horner & Wagner, 1992); Begonia sp. (Begoniaceae),solitary prisms (Horner & Zindler-Frank, 1981) and Begonia maculata , B. manicata , B. metallica , solitary prisms and druses(Al-Rais et al., 1971); Coleus sp. (Labiatae), solitary prisms anddruses (Al-Rais et al., 1971) and Beta vulgaris (Chenopodiaceae),solitary prisms and cylindrics (Al-Rais et al., 1971); and, Echin- omastus intertextus , Echinocactus horizonthalonius , Escobaria tuberculosa (Cactaceae), druses (Rivera, 1973). Reports on intra-cellular COD in plants using crystal morphology as the only criterion include: Telfairia sp., solitary prisms (Okoli & McEuen,1986); Acacia senegal , solitary prisms (Parameswaran & Schultze,1973); and, Aglaonema modestum , Hydrosome rivieri , solitary prisms (Genua & Hillson, 1985).

In addition to periplasmic COM deposits (Pennisi et al.,

2000), D. sanderiana forms crystals in intracellular locations.Their chemical, crystallographic, and ultrastructural aspectsare the focus of this paper.

Materials and Methods

Intracellular crystal extraction and processing

Individual raphides were extracted by pressing freshly cutleaves of Dracaena sanderiana hort Sander ex M.T. Mast.(Dracaenaceae) onto circular glass coverslips and glass slides.Samples were analysed with a scanning electron microcope and

X-ray powder diffractometer as outlined below. Intracellulardeposits other than raphides were obtained from two sources,mature and immature leaves. Pieces from mature leaves andbasal portions of leaf primordia (5–10 mm in length) wereplaced in a maceration solution containing cellulase (1.0% w/v),hemicellulase (1.0% w/v), and pectinase (0.1% w/v) (ProtoplastIsolation Enzyme Solution I, Sigma (Sigma-Aldrich Company,St. Louis, MO, USA) ) for 24 h. Basal portions were used tominimize contamination from cuticular periplasmic COMcrystals (Pennisi et al., 2000). These portions were cut underan optical microscope equipped with polarizing optics toobserve the cuticular crystals. The maceration procedurereduced all internal tissue to individual cells and the epidermis

to a long tube (due to the shape of monocotyledonous leaf primordia). The epidermis was removed, and the cell suspension

was pipetted onto glass slides and circular glass coverslips. Theglass slides and circular glass coverslips were examined with anoptical microscope and any raphide contamination removed.The suspension was flooded with water, causing protoplastswelling and cell rupture. Intracellular crystals were freed andsettled to the bottom of the suspension. Excess water andcellular debris were drawn off with filter paper. Three waterrinses were followed by three 100% ethanol rinses. Coverslips

were prepared for SEM, and glass slides were processed for X-ray diffraction as outlined elsewhere (Pennisi et al., 2000).Results were compared with American Society for Testing Materials (ASTM) X-ray standards for calcium oxalate mono-hydrate (whewellite) and calcium oxalate dihydrate (weddellite).

ASTM data were obtained from the Joint Committee on PowderDiffraction Standards ( JCPDS) – International Centre forDiffraction Data 1996.

Light and transmission electron microscopy

Procedures for light microscopy (LM) and TEM are describedin detail elsewhere (Pennisi et al., 2000).

Results

Calcium oxalate monohydrate raphides

D. sanderiana leaf primordia develop intracellular crystals,

each containing a centrally located bundle of numerous indi-vidual crystals termed raphides (Fig. 1a–c). High birefringence(Fig. 1b) and X-ray diffraction data (Table 1) confirmed thatthe raphides were composed of COM. The raphides are 80–100 µm long with sharp pointed ends (Fig. 1d) and irregularedges (Fig. 1e). Raphide bundles contain 100–150 individualcrystals (Fig. 1b). Ultrastructurally, the raphide idioblasts exhibitseveral distinctive characteristics. Paracrystalline bodies withclosely spaced subunits were observed (Fig. 2a). Individualraphides are located randomly in the cell vacuole and measuredapprox. 1µm in transverse section. All raphides are embeddedin a mucilagenous matrix different from the surrounding

cytoplasm (Fig. 2b). Individual raphides are orientated randomly lengthwise with respect to one another, with large spacesbetween individual crystals (Fig. 2b–c). The most striking feature of D. sanderiana raphide bundles are the crystal chambers(Fig. 2c–f ). Each crystal is surrounded by a lamellate crystalchamber that is not connected to neighbouring chambers andis distinct from the mucilagenous matrix of the raphidebundle. The chambers have double membrane walls, and loop-like lamellate extensions along their wide ends (in transversesection) (Fig. 2d–f ). The length of the lamellate extensionsranges from 0.5 µm to 1.6 µm, and some extensions appearedto end blindly without completing a full loop (Fig. 2f). Theblind ends probably reflect the plane of sectioning. Some

sections show loop-like extensions connected to only one sideof the chamber wall (Fig. 2f ).

Calcium oxalate dihydrate crystals

Numerous tetragonal crystals are present in immature leaf primordial cells observed under polarized light (Fig. 3a).

When mature leaf mesophyll cells are isolated by macerationvariously sized rod-like as well as some prismatic crystals areevident (Fig. 3b). The rod-like deposits are small (≈ 4–5 µm),




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Fig. 3 Light microscopy (LM) of calciumoxalate dihydrate (COD) crystals, andtransmission electron microscopy (TEM)micrographs of leaf mesophyll cells ofDracaena sanderiana. (a) This cell in the leafprimordium is completely filled with prismaticcrystals (arrow) of low birefringence.(b) Isolated mature cell with numerousintracellular rod-like crystals (right pointingarrows) and a single bipyramid crystal (leftpointing arrow). Bars, 10 µm (c–d) Isolatedrod-like crystals with cross-polarized light.In (d), a first order red λ-plate gives amagenta-coloured background. The darkrod-like crystals and one of the crystalsforming a cross (indicates twinning, seeFig. 5g) are in the extinct ion position (arrow).Bars, 5 µm (e–f) Vacuoles with pronouncedangular outlines (stars) in epidermal (e) andmesophyll (f) cells. CW, cell wall. Vacuolarmembranes are continuous with two profilesof RER (arrows). Bars, 1 µm. CW indicates theposition of the cell wall in the figure.

Fig. 2 Transmission electron microscopy (TEM) micrographs of raphide idioblasts in Dracaena sanderiana leaves. Abbreviations: CW, cell wall;CYT, cytoplasm; PB, paracrystalline body; RH, raphide hole; MB, mucilagenous body, matrix; V, vacuole. (a) Paracrystalline body with closelyspaced subunits. (b) In ultrathin sections crystals are not visible since they are not infiltrated by the resin and fall out during sectioning; however,their original locations remain visible as white ‘holes’. The boundary between the cytoplasm and mucilagenous body is arrowed. (c) Portion ofraphide bundle in a developing leaf cell. Note the larger spaces between the individual raphides compared with (f), and the irregular orientationof crystals with respect to each other. The loop-like crystal chamber extensions do not appear to be connected with each other, and do notextend to the edge of the mucilagenous body (down arrow). Note also the blind end of the crystal chamber extension (up arrow). (d) The edgesof the chamber walls can be discerned (right pointing and down black arrows) and in some places appear to connect to the dark extensions atthe corners as well as some of the sidewalls (left pointing black arrow). Some crystal chamber extensions are symmetrical (up white arrow), whileother are asymmetrical (left pointing white arrow). Bar, 1 µm (e) Joining point of the lamellate extensions (arrow). Bar, 100 nm (f) Two blindends (black arrows) of the loop-like extensions. Within this section one lamellate part of the loop appears not connected to the other lamellatepart (white arrow). Bar, 100 nm. Abbreviations used in the figure are CYT, cytoplasm; RH, raphide hole; and MB, mucilagenous body, matrix.




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Discussion

Calcium oxalate monohydrate raphides

The raphides in D. sanderiana are composed of COM, whichis consistent with previous reports of raphides in D. fragrans (Scurfield and Mitchell, 1973). Mature raphides bundles inD. sanderiana exhibit characteristics typical of System IIcrystal idioblasts as defined by Horner & Wagner (1995). Thissystem is exemplified by the monocotyledonous raphide idioblasts

in Typha , Vanilla and Yucca , and typified by lamellate sheathsaround the chamber walls, mucilage-like material surrounding the developing crystal chambers, and paracrystalline bodies

with closely spaced subunits (Horner & Whitmoyer, 1972; Wattendorff, 1976; Tilton & Horner, 1980). The loop-likeextensions of the crystal chambers in D. sanderiana are very similarto chamber wall extensions of Agave raphides (Wattendorff,1976). However, in Agave these structures are larger, display multiple lamellae and form symmetrical, closed loops, whilein D. sanderiana some of the raphide chamber extensions aresingle lamellae, and are less symmetrically orientated than theloop-like extensions in Agave raphide chambers. Unlike thecrystal lamellae in Typha , which are continuous with lamellae

from neighbouring crystals (Horner et al., 1981), the chamber

lamellae in D. sanderiana do not appear to anastomose withother crystal chambers and do not show any discerniblecontinuity with the vacuolar membrane (tonoplast). Theparacrystalline body (Fig. 3a) is an enigmatic structure, whichis rarely observed, and no satisfactory explanation of its natureand function has been determined (Barnabas & Arnott, 1990).It has been hypothesized as a raphide precursor (Horner &

Wagner, 1995).Development of raphide crystal idioblasts has been extens-

ively documented (Franceschi & Horner, 1980). Theoriesconcerning the development of these crystal morphologies arenumerous, and some theories have implicated macromolecules(i.e. proteins and complex polysaccharides) (Webb et al., 1995;

Webb, 1999). Crystal chambers may act as molds and controlboth the shape and size of the crystals within them (Arnott,1976). A wide variety of additives have altered COM morpho-logy in vitro and produced crystals resembling some of thosefound in plants (Cody & Horner, 1984; Cody & Cody, 1987;Stevens et al., 1999). Arnott’s suggestion that the crystal cham-

bers may act as molds controlling the crystal shape has beenextended by one hypothesis of how macromolecules (acidicproteins) can affect the mineral phase via a polymer-inducedliquid precursor (PILP) process (Gower & Odom, 2000).Nonequilibrium crystal morphologies were generated in a solution crystallization of calcium carbonates in the presenceof polyaspartic acid. This strongly acidic polypeptide induceda liquid phase separation, in which droplets of a liquid pre-cursor to the mineral accumulated in the form of mineralfilms and coatings. As the precursor is a liquid, it can fill a space,and the final mineral retains the shape of the precursor ‘molded’to form the unusual morphologies (Gower & Odom, 2000).

The PILP process has recently been demonstrated in experi-ments with CO (Malpass & Gower, 1999).

Calcium Oxalate Dihydrate crystals

Intracellular COD crystals in D. sanderiana exhibit two typesof morphology. One is typical of the tetragonal-bipyramidalclass, with expression of {101} faces enclosing two tetragonalpyramids at both crystal ends. The other crystal morphology is atypical of the tetragonal class. Development of someunexpected {100} faces enclosing the tetragonal prism wasobserved. This crystal form (combination of a tetragonaldipyramid and a tetragonal prism) has been documented in

Begonia (Horner & Zindler-Frank, 1981) and Capsicum

Ta e 2 Comparison o American Society or Testing Minera s(ASTM) data of calcium oxalate dihydrate and intracellular crystalsextracted from the mesophyll of Dracaena sanderiana

ASTM weddellitex CaC2O4·2H2O Dracaena sanderiana

D, Åy I/I0z D, Å I/I0

*6.18w 100 6.21 100*4.42 30 5.99 25*3.78 65 3.72 16.52.41 16 2.41 20.32.24 25 2.25 13.91.90 16 – –

xASTM data were obtained from Joint Committee on PowderDiffraction Standards (JCPDS) – International Centre for DiffractionData 1996. yD is the wavelength spacings in Ångstroms. zI/Io is relativeintensity of diffraction response compared to the primary peak.wThe three major peaks are indicated by an asterisk (*) in eachanalysis.

Fig. 4 Scanning electron microscopy (SEM) micrographs of intracellular crystals isolated from leaf primordia of Dracaena sanderiana showingtypical (a–c) and atypical (d–h) calcium oxalate dihydrate (COD) morphology. (a) This COD crystal has one four-fold axis of symmetry resultingin tetragonal pyramids at both crystal ends. Arrow indicates the plane (100) enclosing a prism. (b) This twinned COD is a rotational combination,showing both the tetragonal bipyramid (black arrows) and the tetragonal prism (white arrow). (c) An interpenetrant twinned crystal shows ahigh degree of defects, including holes on the pyramidal surface (arrow). (d–f) Crystals display {111} faces (black stars) and {110} faces, whichare inconsistent with the typical bipyramidal COD morphology (compare with (a)). The pinacoid {001} is present. (g) The {101} faces (arrows)are small compared to the typical COD morphology (compare with (a)). The crystals also exhibit somewhat rounded corners (white stars). Bars,1 µm.




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(Wagner, 1983; Horner & Wagner, 1992). To the best of ourknowledge, prior to this study, detailed identification of CODcrystal faces has not been attempted nor have the unusualcrystal morphologies in D. sanderiana COD crystals resulting

from expression of {001}, {111}, and {110} faces beenpreviously documented.

Fig. 5 represents a hypothetical sequence, which helpsexplain the changes in crystal COD morphology, which wereobserved in D. sanderiana intracellular crystals. All variationsobserved in D. sanderiana COD crystals (Fig. 4b–h) can bederived from the typical morphology in (a) by the develop-ment of some additional faces (b–g). The typical tetragonalbipyramid of synthetic COD crystals (a) is achieved by develop-ment of {101} faces. However, when the {100} face develops,

a new crystal shape (b) emerges (compare with Fig. 4a).Furthermore, development of the terminal pinacoid plane{100} and the {111} planes (f ) results in yet another crystalshape similar to those in Fig. 4(d,f,h). Development of a set of

{110} planes (e) results in a shape similar to the crystals inFig. 4(g–h). The rod-like crystals observed with light micro-scopy (Fig. 3b–d) are similar to the twinned COD crystalsillustrated by Frey-Wyssling (1981). Their morphology couldbe explained by the relative expression of {100} faces com-pared to the typical {101} faces (g ). The planes observed in D.sanderiana COD crystals are not consistent with the syntheticCOD morphology. Instead, they are unstable, high-energy crystal faces, commonly developed in crystals precipitated in vitro in the presence of various solution constituents (Addadi

Fig. 5 Schematic illustration showinghypothetical growth modifications of crystalfaces in intracellular calcium oxalate dihydrate(COD) crystals in Dracaena sanderiana.(a) Typical tetragonal COD bipyramids grownin vitro with no additives. (b) Developmentof tetragonal prisms {100} in intracellularCOD crystals in D. sanderiana (compare withFig. 3b). (c) Development of {001} pinacoids.(d) Growth of the {001} pinacoid planes isstabilized resulting in large {001} faces andtruncated {101} faces (compare with Fig. 4d).

(e) Additional habit modifications withdevelopment of {111} and {110} faces(compare with Fig. 4). (f) Same as (e) vieweddown the c axis. (g) Twinning of COD crystalsresulting in appearance of ‘crosses’ (comparewith Fig. 4c–d). Note the large area of the{100} faces compared to {101} faces.




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& Weiner, 1989). Development of less stable crystal faces canoccur due to interactions with solution constituents (i.e.acidic macromolecules, including proteins extracted frombiominerals). Crystal habit modifications of COM have beenachieved by the growth of crystals in the presence of citrateand phosphocitrate (Sikes & Wierzbicki, 1996). In addition,alterations of the less common COD crystals have beenbrought about by growth of crystals in solutions with α,ω-dicarboxylic acids (Stevens et al., 1999). Suberic acid was alsosuggested to have a structural motif, which matched the {110}face of COD thus stabilizing expression of these high-energy planes.

The intracellular vacuoles observed in D. sanderiana withtheir angular outlines may have contained the COD crystals(Fig. 3e–f ). Crystals are not preserved in ultrastructural prep-arations, but one indication of their presence is the spaces they previously occupied. If association between the vacuoles andthe crystals is accepted, several implications follow. Since thevacuoles were attached to parent RER membranes, the COD

precipitation inside D. sanderiana cells may be controlled by the elaboration of RER crystal vacuoles. Further support of this hypothesis is that crystal morphology is modified by thedevelopment of unstable crystal faces, presumably throughinteractions with impurities in the vacuolar medium. Thedefects present in some crystals (holes and cracks on the crystalsurface, Fig. 4c) are also typical of biogenically precipitatedminerals, as are rounded crystal corners (Fig. 4g–h), which areattributed to non-specific interactions with impurities (Addadi& Weiner, 1989). Our extraction procedure involved enzymatictissue digestion to release the crystals, therefore these defects

were not likely artifacts. Space delineation is one of the most

distinctive features of biologically controlled biomineraliza-tion, and lipid membranes are the most common way of seal-ing off a predetermined compartment (Lowenstam & Weiner,1989). This sealing off process allows selective uptake of ionsand provides a means to control concentration and composi-tion of the initial solution from which the mineral forms.

Our study of crystalline deposits in D. sanderiana leads tothe following conclusions. There is definitive evidence for twohydrate forms of CO, COM and COD, in the same plantspecies. Three distinctive crystal morphologies exist, periplasmicCOM crystals, intracellular COM raphides, and intracellularCOD crystals. The factors controlling CO phase and morphology in D. sanderiana remain to be determined, but the constancy

of CO forms in tissue-specific locations seems to indicatea highly developed phytosystem for biologically controlledbiomineralization.D. sanderiana has proven to be an excellentexample of a phytosystem with highly controlled depositionof biogenic CO hydrates.

Acknowledgements

The authors thank Drs Karen Koch and Bart Schutzman forreview of the manuscript and instructive criticism.

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