Turk J Biol 36 (2012) 386-393 © TÜBİTAK doi:10.3906/biy-1109-35

Calcium oxalate crystal types in three species (Quercus L.) in

Bedri SERDAR1, Hatice DEMİRAY2 1Karadeniz Technical University, Faculty of Forestry, Department of Forest Botany, Trabzon - TURKEY 2Ege University, Faculty of Science, Department of Botany, İzmir - TURKEY

Received: 30.09.2011 ● Accepted: 17.02.2012

Abstract: Crystal types from 3 oak species representing 3 diff erent sections of the Quercus were identifi ed. Crystals were studied with scanning electron microscopy aft er localisation by light microscopy. Th e crystals were composed of oxalate silicates as (calcium oxalate monohydrate) composites. In Quercus macranthera Fisch. et Mey. subsp. syspirensis (C.Koch) Menitsky (İspir oak) from the white (section Quercus: Leucobalanus), whewellite and trihydrated weddellites coexisted. Axial parenchyma cells included 30 crystal chains with walls as chambers in uniseriate ray cells; some cells had many crystals without any chambers and were small. Long thin crystals were found adherent to the membrane in the tracheary cell lumens of latewood. Quercus cerris L. var. cerris (hairy oak/Turkey oak) from the red oak group (section Cerris Loudon) is extremely rich in rhomboidal crystals. In species Quercus aucheri Jaub. et Spach (grey pırnal) from the evergreen oak group (section Ilex Loudon) rhomboidal crystals were found in axial parenchyma and multiseriate ray cells. Lignifi ed wall layers were not observed around the crystals with chambers.

Key words: Calcium oxalate silicate, crystals, crystal morphology, Quercus, Turkey

Introduction some Araceae (e.g., Xanthosoma sagittifolium) (2). Most invest considerable resources in In 3 main types of calcium oxalate cytoplasmic inclusions such as starch, tannins, crystal occur: raphides, styloids, and druses. Th e silica bodies, and calcium oxalate crystals in absence of raphides, the most common crystal type some of their cells. Calcium oxalate crystals are in monocots, represents a synapomorphy in groups widespread in fl owering plants, both dicotyledons such as Alismatales, Poales, and some Liliales. Druses and monocotyledons, indicating their importance (sphaeraphides) are common in dicotyledons and in basic processes of growth and development. It relatively rare in monocotyledons, where they are has been proposed that calcium oxalate crystals play mostly restricted to some early-branching taxa such a role in ion balance, defence, tissue support, as Acorus, Araceae, and Tofi eldia (3). Th e control detoxifi cation, and light gathering and refl ection of crystal morphology is a tightly regulated genetic (1). In some plants crystals have more specialised process, and a simple point mutation can drastically functions, such as promoting air space formation alter the size and shape of crystals in legumes (4,5). in aquatic plants or helping to prevent herbivory, Biomineralisation is widespread among although many plants containing calcium oxalate photosynthetic organisms in the ocean, inland crystals are eaten by birds and animals, such as waters, and on land. In biomineralisation the most

386 B. SERDAR, H. DEMİRAY

quantitatively important biogeochemical role of land Anatomical sectioning of wood specimens plants today is silica deposition in vascular plants, Wood samples were boiled in water and stored in 50% especially grasses. Th e form calcium carbonate aqueous ethanol, sectioned using a sliding microtome takes in biominerals, and presumably the evolution of organisms that produce it, have been infl uenced at a thickness of about 20-25 μm, and stained with by abiotic variation in calcium and magnesium safranin O and alcian blue. Sections were washed with concentrations in seawater. Calcium carbonate distilled water aft er staining and transverse, radial, deposition has probably also been infl uenced by and tangential sections were mounted on glycerine- dioxide concentration, which is in part gelatine aft er dehydration through an alcohol series biologically determined. Overall, there has been of 50%, 75%, and 95% (11,12). less biological feedback on substrate availability for calcifi cation than substrate availability for Terminology used is in accordance with the list silicifi cation (6). Opaline silica (the form of silica of microscopic features for hardwood identifi cation deposition by organisms) is undersaturated with (13). respect to dissolved silicic acid in almost all natural Isolation of crystals: scanning electron microscope waters; an exception is silicic acid-rich hot springs (SEM) and powder X-ray diff raction methods of the kind that display excellently preserved fossils from the Rhynie and Windyfi eld cherts of Wood sections were processed according to Horner the Lower Devonian. Th ese are represented today (14), and enzyme solution containing 5% cellulase at Yellowstone National Park (7). Cases of silica and 1% pectolyase was used to aid in the release of deposition involve active transport of silicic acid crystals from their surrounding cells and crystal across one or more biological membranes. A possible walls. Th in wood sections were coverslipped with exception is extracellular precipitation of silica in Permount on slides and examined using a JEOL JSM- vascular plants, where the concentration of xylem sap by transpiratory water loss from the shoots may 6060 SEM operated at 10 kV; MAG 700 was used to lead to supersaturation of monomeric silicic acid in visualise isolated crystals in the woods. Specimens the apoplasm. However, in cases of substantial silicic were sputter-coated with gold, and secondary acid deposition within vascular plants there is active electrons were recorded. Images were photographed. transport of silicic acid somewhere between the soil Th in sections of wood with isolated crystals were solution and the xylem sap (8-10). attached to a glass slide with Vaseline, and X-ray In this study, crystal types of 3 endemic oaks: diff raction analysis was performed with a Jeol JSDX- Quercus macranthera Fisch. et Mey. subsp. syspirensis 100 S4 X-ray diff ractometer apparatus using Cu X-ray (C.Koch) Menitsky (İspir oak) (section Quercus: tubes and a Ni fi lter in 32 kV, 22 mA. Th e diff raction Leucobalanus), Quercus aucheri Jaub. et Spach (grey pattern was compared with American Society for pırnal) (section Ilex: Loudon), and Quercus cerris L. Testing and Materials (ASTM) data for the hydration var. cerris (hairy oak/Turkey oak) (section Cerris: forms of Ca oxalate. Loudon) were identifi ed by X-ray, FT-IR analysis, and microscopic observations for the fi rst time. Part of the wood sample was crushed into fi ne powder for analysis. Th e powdered sample was homogenised in spectroscopic grade KBr (1/20) Materials and methods with an agate mortar and pressed into 3 mm Wood materials of this study were obtained from pellets with a hand press. Th e infrared spectra were stems of 3 Quercus taxa. Th e samples of Quercus acquired using a Perkin-Elmer spectrum 100 FT-IR macranthera Fisch. et Mey. subsp. syspirensis (C.Koch) Menitsky, Quercus cerris L. var. cerris, and spectrophotometer at Ege University Textile Faculty, Quercus aucheri Jaub. et Spach were collected from İzmir, at a resolution of 4 cm–1. Th e spectra were taken Yozgat (130 m), Kastamonu (40 m), and Muğla (400 in the 400-4000 cm–1 region, and room temperature m), respectively. was 20 °C during the analysis.

387 Calcium oxalate crystal types in three oak species (Quercus L.) in Turkey

Results Some of the Quercus macranthera et Mey. subsp. syspirensis specimens gathered at diff erent sites (Boyabat and Yozgat) in Turkey were rich in crystals. Crystals were rare or absent in the wood specimens gathered from the Kastamonu and Sivas districts. Crystals were not present inside the multiseriate a b ray cells. Crystals were observed inside the axial parenchyma, uniseriate ray cells, and the vessel elements of late wood. Crystal shapes were tetragonal, and also kinked and straight, in Q. macranthera subsp. syspyrensis (Figure1a). While long chambered crystal chains occurred in axial parenchyma that generally have 30 cells, very compact crystals were commonly found in non-chambered cells. In addition, long, c thin, needle-like crystals were observed in the lumens of the vessel elements of latewood adherent to the cell wall (Figure 1b). Prismatic crystals were common in uniseriate ray cells in Q. cerris var. cerris although they were very small. Specimens from the north of Turkey had sparse crystals, and crystal quantity increased in the from more southern provenances. Multiseriate ray cells were especially rich in crystals. Chambered crystals occurred in long chains in axial parenchyma cells. Starch grains were d seen inside the ray cells and axial parenchyma cells of specimens gathered from southern Turkey (Figures Figure 1. Q. macranthera subsp. syspyrensis (a-b); a: crystals in 1c, d). axial parenchyma cells, b: crystals in vessel element. Q. cerris var. cerris (c-d); c and d: crystals in multiseriate Quercus aucheri Jaub. et Spach was rich in rays. Scale bars (a-d) = 50 μm. crystals; rhomboidal crystals were found in the axial parenchyma and ray cells. Rhomboidal crystals were the Si-O-Si bending vibration region (400-700 cm–1) tetragonal. Lignifi ed integuments, however, were of quartz, the band at 695 cm–1 determines whether indistinct or absent from crystalliferous ray cells it is crystalline or amorphous (13). In the amorphous (Figures 2a-e). state this band will be missing. Th e absorption Analysis of the crystals by X-ray diff raction band at 695 cm–1 is due to vibrations in octahedral indicated that calcium oxalate was present as site symmetry, at 780 cm–1 it is due to vibrations in silicates in the taxa of 3 diff erent sections. Th e FTIR tetrahedral site symmetry (16,17), and 781 cm–1 is spectrum revealed several absorption bands at 608- present only in Q. macranthera. Tetrahedral symmetry 897, 1046-1739, and 2346-3436 cm−1. In Q. cerris L. is stronger than octahedral symmetry. Th erefore, var. cerris and Q. aucheri amorphous silica exhibited in any structural change, the damage occurs fi rst in a relatively strong peak at 800 nm, separate from octahedral and then in tetrahedral symmetry. When the band of crystalline silicate (15). Th e structure the temperature was rapidly brought to 600-700 of most polymorphous SiO2, both crystalline and °C, the SiO4 tetrahedral could not be ordered into a amorphous, is based on a tetrahedral unit of silicon crystalline state and was preserved in an amorphous coordinated to 4 oxygen atoms. Amorphous silica is silicate phase. Th e carbonate structure contains -2 one of the polymorphous forms of silica, and at high an isolated CO 3 group with a doubly degenerate temperatures it can easily transform from quartz. In symmetric stretch (ν3) in the 1507-1599 cm–1 region

388 B. SERDAR, H. DEMİRAY

abcd

e

Figure 2. Q. aucheri; a-d: twinned crystals in axial parenchyma and ray parenchyma cells, e: pits between ray and vessel. Scale bars (a-e) = 50 μm.

(18). Th e absorption bands at 3420, 3429, and 3436 was found to be the only crystalline phase present in cm–1 and 1624, 1633, and 1644 cm–1 arise due to the CaOx (Figure 3a), and the composite was obtained in adsorbed water molecules commonly observed in all 3 species of Quercus (Figures 3b, c) (19). Th e OH natural silica (17). Th e XRF results show that the stretching of coordinated COM water molecules was major component of the sample is SiO2. Th e broad demonstrated by broad absorption bands between Bragg peaks observed in X-ray diff raction studies 3000 and 3500 cm−1 in all samples. Th e water reveal that the sample could be nanocrystalline calibration bands of the composites (660 and 584 quartz. Figure 3 shows the XRD spectra of CaOx cm−1) were shift ed slightly so that they were lower −1 and CaOx-SiO2 composites formed in water. COM than those of CaOx (667 and 594 cm ). Th is was

389 Calcium oxalate crystal types in three oak species (Quercus L.) in Turkey

probably due to an increase in the number of water of calcium oxalate occur naturally as 3 molecules in the (i.e. the presence species: whewellite (monohydrate, known from of COT) (20). Th is observation was in contrast to the some beds), (dihydrate), and a very results of XRD and SEM imaging in which COT was rare trihydrate called caoxite (24). Macrocrystals of the main component of the composite formed only the polyhydrate were found in the fossil deposits of in Q. macranthera taxa. Due to the Si-O-Si stretching the Weddell Sea and named weddellite. Its crystal vibration of amorphous silica, another weak band structure was disclosed in a kidney calculus and is −1 isomorphic with tetragonal strontium oxalate. was present at 1113 cm in the CaOx-SiO2 composite deposit spectrum (Figure 3d) (21). Th e other 2 taxa, Th e evolutionary background of silicifi cation −1 Q. aucherii and Q. cerris var. cerris, have 608 cm demonstrates that diatoms, which have molecular bands of composite characteristics. SEM imaging of genetic similarities among the silicic acid transporters composite materials in Q. macranthera wood (Figures of these organisms (ochristian algae), were known 3d-h) showed a mixture of hexagonal platelets of to be the single origin of silicifi cation; however, COM and needles of COT. Aggregates of monoclinic there is no sequence similarity among the 2 silicic COM crystals, with contact twinning [attachment acid transporters known from Oryza sativa and of 2 individual crystals along (1 0 0) plane] being those known from ochristian algae (25-27) or the more prevalent in the binary mixture (Figure 3d). putative transporter from sponge (28). Among the FT-IR was used to analyse the molecular structure vascular plants, Poales and Equisetales have a shared of sample components. Th e IR spectra of CaOx and hemicellulose component that could be a template for composites exhibited peaks around 1628 cm−1, 1317 silica deposition, although they are phylogenetically cm−1 (symmetrical and antisymmetrical oxalate distant within the euphyllophyte plants (29,30). C=O bond stretching), 781 cm−1 (O-C-O in-plane For terrestrial plants there is no signifi cant fossil bending), and 517 cm−1 (O-C-O in-plane rocking), evidence on which to base the environment- which is characteristic of COM (20). evolution interactions in the production of alkaline earth biominerals such as calcium oxalate, despite both silica and calcium carbonate deposition in the Discussion ocean throughout the Phanerozoic. However, the Th e presence of CaOx crystals as composites in impact of mineralising photosynthetic organisms the 3 diff erent Quercus taxa represents important on the availability of soluble substrates for phylogenetic, systematic, and evolutionary evidence. mineralisation for calcifi ers has been smaller than Th ese structures are formed as biominerals of the impact for silifi ers (31). Lauraceae and , diff erent morphologies (raphides, prisms, druses, tropical plants that have fossils from the Cretaceous, and sand) and silicon dioxide bodies, which are provide an example of the eff ect of environmental taxon-specifi c and heritable (22). Silica accumulation factors (32). Th e wood anatomy of some Lauraceae was especially prevalent in (Araceae, from the Amazonian forest and the Atlantic forest Commelinales, Poales, and Hanguana) and Orchids of south-eastern demonstrate the occurrence (23). In Quercus species, whewellite (Ca oxalate of silica bodies in ray cells (33). Among the diff erent monohydrate) prismatic crystals were observed; Quercus taxa, Q. macranthera must be later than only in Q. macranthera were weddellite (Ca the other Quercus taxa phylogenetically because oxalate trihydrate) crystals found together with it contains whewellite and Ca oxide (Ca oxalate composite whewellite forms. When we investigated trihydrate) together inside the plant. Elsewhere in taxonomically and phylogenetically, the coexistence the plant kingdom a few observations have been of the needle-shaped trihydrate and the monohydrate reported. Opuntia fi cus-indica for example, has forms of calcium oxalate inside the same plant cell highly productive crassulacean acid metabolism demonstrated synapomorphy among the Quercus (CAM). In the parenchymal tissues of Opuntia fi cus- taxa; however, the occurrence of silicates as calcium indica (Miller) 2 forms of calcium oxalate that diff er oxalate composites show symplasty between the in hydration level were identifi ed (34). In a study monocots and Quercus taxa. Hydrated forms conducted with sessile oak wood, crystals were

390 B. SERDAR, H. DEMİRAY

e f

g h

Figure 3. X-ray diff raction patterns of Q. macranthera (a), Q. cerris (b), and Q. aucherii (c); FT-IR analysis patterns of 3 Quercus taxa (d); SEM micrographs of Q. macranthera crystals (e-f), Q. cerris crystals (g), and Q. aucherii crystals (h).

391 Calcium oxalate crystal types in three oak species (Quercus L.) in Turkey

found in sapwood rings. Th e X-ray negative method crystals were observed in the uniseriate ray cells, indicated that trihydrated calcium oxalate and silica vessels, vasicentric tracheids, or fi bres of sessile oak reach peak concentrations at the beginning of the wood. late wood zone of the annual ring and mainly in multiseriate rays (35). While the results from sessile oak are similar to those found in Quercus taxa, Acknowledgements one diff erence emerged. In addition to crystalloid We want to express our special thanks to Dr. Pieter fragments, amorphous granules containing 66% Baas for his valuable guidance and critical reading of iron, 0.65% sulphur, 0.59% calcium, and 0.57% the manuscript. A condensed version of this article silicium were found in sessile oak wood. Amorphous was presented at the First International Symposium granules were not found in the investigated Quercus on Turkish-Japanese Environment and Forestry (4-6 taxa. However, chambered crystal-bearing cells in November 2010). multiseriate rays were a characteristic of sessile, hairy, and grey pırnal oaks and these were rhomboidal Corresponding author: shaped in the latter 2 oaks. In addition, prismatic crystals have been found in axial parenchyma cells Bedri SERDAR inside of or adjacent to multiseriate rays, mostly Karadeniz Technical University, in isodiametric or slightly elongated form. In the Faculty of Forestry, taxa investigated for this study, uniseriate ray cells (İspir oak), vessels, and vasicentric tracheids (hairy 61080 Trabzon-TURKEY oak) had long crystal clusters like sand crystals; no Email: [email protected]

References

1. Franceschi VR, Horner HT. Calcium oxalate crystals in plants. 10. Kutlu N, Terzi R, Tekeli Ç et al. Changes in anatomical structure Bot Rev 46: 361-427, 1980. and levels of endogenous phytohormones during leaf rolling in Ctenanthe setosa under drought stress. Turk J Biol 33: 115-122, 2. Walter WG, Khanna PN. Chemistry of the aroids. I. 2009. Dieff enbachia seguine, amoena and picta. Econ Bot 26: 364-372, 1972. 11. Ives, E. A Guide to Wood Microtomy, Sproughton, Nijhoff / Junk Publishers, 114 p, 2001. 3. Prychid CJ, Rudal PJ. Calcium oxalate crystals in monocotyledons: a review of their structure and systematics. 12. Gerçek, Z. Türkiye’de Yetiştirilen Camellia sinensis (L.) Ann Bot-London 84: 725-739, 1999. Kuntze’nin İç Morfolojik Özellikleri ve Farklı Yetişme Koşullarının Bu Özellikler Üzerine Etkisi (PhD Th esis), K.Ü. 4. Nakata PA. Calcium oxalate crystal morphology. Trends Plant Basımevi, Trabzon, 1984. Sci 7: 324, 2002. 13. IAWA Committee. IAWA list of microscopic features for 5. Sarghein SH, Carapetian J, Khara J. Th e eff ects of UV radiation hardwood identifi cation. IAWA Bull 10: 221-332,1989. on some structural and ultrastructural parameters in pepper (Capsicum longum A.DC.) Turk J Biol 35: 69-77, 2011. 14. Horner HT, Zindler FE. Calcium oxalate crystals and crystal cells in the leaves of Rhynchosia caribaea DC. (Leguminosae- 6. Raven JA, Giordano M. Biomineralization by photosynthetic Papilionoideae). Protoplasma 111: 10-18, 1982. organisms: Evidence of coevolution of the organisms and their environment. Geobiology 7: 140-154, 2009. 15. Ojima J. Determining of crystalline silica in respirable dust samples by infrared spectrophotometry in the presence of 7. Channing A, Edwards D. Experimental taphonomy: interferences. J Occup Health 45: 94-103, 2003. silicifi cation of plants in Yellowstone hot-spring environments. Transactions of the Royal Society of Edinburgh 94: 503-521, 16. Schneider H. Shock-induced thermal transformations of Ries- 2004. biotites. Contributions to Mineralogy and Petrology, 43: 233- 243, 1974. 8. Raven JA. Th e transport and function of silicon in plants. Biol Rev 58: 179-207, 1983. 17. Parthasarathy G, Kunwar AC, Srinivasan R et al. Occurrence of moganite-rich chalcedony in Deccan fl ood basalts, Killari, 9. Raven JA. Silicon transport at the cell and tissue level. In Silicon Maharashtra, India. Eur J Mineral 13: 127, 2001. in Agriculture. Elsevier, Amsterdam 8: 41-55, 2001.

392 B. SERDAR, H. DEMİRAY

18. Parthasarathy G, Chetty TRK, Haggerty SE et al. Th ermal 28. Schroder HC, Perovic-Ottstadt S, Rothenberger M et al. stability and spectroscopic studies of zemkorite: Acarbonate Silica transport in the demosponge Suberites domuncula: from the Venkatampallekimberlite of southern India. Am fl uorescence emission analysis using the PDMO probe and Mineral. 87: 1384, 2002. cloning of a potential transporter. Biochem J 381: 665-673, 2004. 19. Kontoyannis CG, Bouropoulos NC, Koutsoukos PG et al. Urinary stone layer analysis of mineral components by Raman 29. Fry SC, Nesselrode BHWA, Miller JG et al. Mixed-linkage spectroscopy, IR spectroscopy, and X-ray powder diff raction: a (1→3, 1→4)-β-D-glucan is a major hemicellulose of Equisetum comparative study. Appl Spectrosc 51: 1205-9, 1997. (horsetail) cell walls. New Phytol 179: 104-114, 2008. 20. Babic-Ivancic V, Furedi-Milhofer H, Purgaric B et al. 30. Sorenson I, Pettolino F, Wilson SM et al. Mixed-linkage Precipitation of calcium oxalates from high ionic strength (1→3, 1→4)-β-D-glucan is not unique to the Poales and is an solutions III. Th e infl uence of reactant concentrations on the abundant component of Equisetum arvense cell walls. Plant J 54: 510-521, 2008. properties of the precipitates. J Cryst Growth 71: 655, 1985. 31. Raven JA, Giordano M. Biomineralization by photosynthetic 21. Bellamy LJ. Th e Infrared Spectra of Complex Molecules, organisms. Evidence of coevolution of the organisms and their Chapman & Hall Ltd, vol 1,3. London. 1975. environment. Geobiology 7: 140-154, 2009. 22. Prychid CH, Rudall PJ, Gregory M et al. Systematics and 32. Kole C. Genome mapping and molecular breeding in plants. biology of silica bodies in monocotyledones. Bot Rev 69: 377- Forest Trees. P. 162, Springer, 2007. 440, 2004. 33. Callado CH, Costa CG. Wood anatomy of some Anaueria and 23. Moller JD, Rasmussen H. Stegmata in Orchidales: character Beilschmiedia species (Lauraceae) IAWA J 18: 247-2 59. 1997. state distribution and polarity. Bot J Linn Soc 89: 53-76, 1984. 34. Malainine ME, Dufresne A, Dupeyre D et al. First evidence for 24. Wyssling AF. Crystallography of the two hydrates of crystalline the presence of weddellite crystallites in Opuntia fi cus-indica calcium oxalate in plants. Am J Bot 68: 130-141, 1981. parenchyma. Zeitschrift für Naturforschung 58: 812-816, 2003. 25. Ma JF, Yamaji N. Silicon uptake and accumulation in higher 35. Vansteenkiste D, Acker J Van, Stevens M et al. Composition, plants. Trends Plant Sci 11: 392-397, 2006. distribution and supposed origin of mineral inclusions in 26. Th amatrakoln K, Hildebrand M. Silicon uptake in diatoms sessile oak wood – consequences for microdensitometrical revisited: a model for saturable and nonsaturable uptake analysis. Ann Forest Sci 64: 11-19, 2007. kinetics and the role of silicon transporters. Plant Physiol 146: 1397-1407, 2008. 27. Yamaki N, Mitatni N, Ma JF et al. A transporter regulating silicon distribution in rice shoots. Plant Cell 20: 1381-1389, 2008.

393