Calcium oxalate crystal types in three oak species

386

Turk J Biol

36 (2012) 386-393

© TÜBİTAK

doi:10.3906/biy-1109-35

Calcium oxalate crystal types in three oak

species (Quercus L.) in Turkey

Bedri SERDAR1, Hatice DEMİRAY

2

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 genus Quercus were identifi ed. Crystals

were studied with scanning electron microscopy aft er localisation by light microscopy. Th e crystals were composed of

calcium oxalate silicates as whewellite (calcium oxalate monohydrate) composites. In Quercus macranthera Fisch. et

Mey. subsp. syspirensis (C.Koch) Menitsky (İspir oak) from the white oaks (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

Most plants invest considerable resources in cytoplasmic inclusions such as starch, tannins, silica bodies, and calcium oxalate crystals in some of their cells. Calcium oxalate crystals are widespread in fl owering plants, both dicotyledons and monocotyledons, indicating their importance in basic processes of growth and development. It has been proposed that calcium oxalate crystals play a role in ion balance, plant defence, tissue support, detoxifi cation, and light gathering and refl ection (1). In some plants crystals have more specialised functions, such as promoting air space formation in aquatic plants or helping to prevent herbivory, although many plants containing calcium oxalate crystals are eaten by birds and animals, such as

some Araceae (e.g., Xanthosoma sagittifolium) (2). In monocotyledons 3 main types of calcium oxalate crystal occur: raphides, styloids, and druses. Th e absence of raphides, the most common crystal type in monocots, represents a synapomorphy in groups such as Alismatales, Poales, and some Liliales. Druses (sphaeraphides) are common in dicotyledons and relatively rare in monocotyledons, where they are mostly restricted to some early-branching taxa such as Acorus, Araceae, and Tofi eldia (3). Th e control of crystal morphology is a tightly regulated genetic process, and a simple point mutation can drastically alter the size and shape of crystals in legumes (4,5).

Biomineralisation is widespread among photosynthetic organisms in the ocean, inland waters, and on land. In biomineralisation the most

B. SERDAR, H. DEMİRAY

387

quantitatively important biogeochemical role of land plants today is silica deposition in vascular plants, especially grasses. Th e form calcium carbonate takes in biominerals, and presumably the evolution of organisms that produce it, have been infl uenced by abiotic variation in calcium and magnesium concentrations in seawater. Calcium carbonate deposition has probably also been infl uenced by carbon dioxide concentration, which is in part biologically determined. Overall, there has been less biological feedback on substrate availability for calcifi cation than substrate availability for silicifi cation (6). Opaline silica (the form of silica deposition by organisms) is undersaturated with respect to dissolved silicic acid in almost all natural waters; an exception is silicic acid-rich hot springs of the kind that display excellently preserved fossils from the Rhynie and Windyfi eld cherts of the Lower Devonian. Th ese are represented today at Yellowstone National Park (7). Cases of silica deposition involve active transport of silicic acid across one or more biological membranes. A possible exception is extracellular precipitation of silica in vascular plants, where the concentration of xylem sap by transpiratory water loss from the shoots may lead to supersaturation of monomeric silicic acid in the apoplasm. However, in cases of substantial silicic acid deposition within vascular plants there is active transport of silicic acid somewhere between the soil solution and the xylem sap (8-10).

In this study, crystal types of 3 endemic oaks: Quercus macranthera Fisch. et Mey. subsp. syspirensis (C.Koch) Menitsky (İspir oak) (section Quercus: Leucobalanus), Quercus aucheri Jaub. et Spach (grey pırnal) (section Ilex: Loudon), and Quercus cerris L. var. cerris (hairy oak/Turkey oak) (section Cerris: Loudon) were identifi ed by X-ray, FT-IR analysis, and microscopic observations for the fi rst time.

Materials and methods

Wood materials of this study were obtained from stems of 3 Quercus taxa. Th e samples of Quercus macranthera Fisch. et Mey. subsp. syspirensis (C.Koch) Menitsky, Quercus cerris L. var. cerris, and Quercus aucheri Jaub. et Spach were collected from Yozgat (130 m), Kastamonu (40 m), and Muğla (400 m), respectively.

Anatomical sectioning of wood specimens

Wood samples were boiled in water and stored in 50%

aqueous ethanol, sectioned using a sliding microtome

at a thickness of about 20-25 μm, and stained with

safranin O and alcian blue. Sections were washed with

distilled water aft er staining and transverse, radial,

and tangential sections were mounted on glycerine-

gelatine aft er dehydration through an alcohol series

of 50%, 75%, and 95% (11,12).

Terminology used is in accordance with the list

of microscopic features for hardwood identifi cation

(13).

Isolation of crystals: scanning electron microscope

(SEM) and powder X-ray diff raction methods

Wood sections were processed according to Horner

(14), and enzyme solution containing 5% cellulase

and 1% pectolyase was used to aid in the release of

crystals from their surrounding cells and crystal

walls. Th in wood sections were coverslipped with

Permount on slides and examined using a JEOL JSM-

6060 SEM operated at 10 kV; MAG 700 was used to

visualise isolated crystals in the woods. Specimens

were sputter-coated with gold, and secondary

electrons were recorded. Images were photographed.

Th in sections of wood with isolated crystals were

attached to a glass slide with Vaseline, and X-ray

diff raction analysis was performed with a Jeol JSDX-

100 S4 X-ray diff ractometer apparatus using Cu X-ray

tubes and a Ni fi lter in 32 kV, 22 mA. Th e diff raction

pattern was compared with American Society for

Testing and Materials (ASTM) data for the hydration

forms of Ca oxalate.

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)

with an agate mortar and pressed into 3 mm

pellets with a hand press. Th e infrared spectra were

acquired using a Perkin-Elmer spectrum 100 FT-IR

spectrophotometer at Ege University Textile Faculty,

İzmir, at a resolution of 4 cm–1. Th e spectra were taken

in the 400-4000 cm–1 region, and room temperature

was 20 °C during the analysis.

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

388

a b

c

d

Figure 1. Q. macranthera subsp. syspyrensis (a-b); a: crystals in

axial parenchyma cells, b: crystals in vessel element. Q.

cerris var. cerris (c-d); c and d: crystals in multiseriate

rays. Scale bars (a-d) = 50 μm.

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 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, 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 trees 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 seen inside the ray cells and axial parenchyma cells of specimens gathered from southern Turkey (Figures 1c, d).

Quercus aucheri Jaub. et Spach was rich in crystals; rhomboidal crystals were found in the axial parenchyma and ray cells. Rhomboidal crystals were tetragonal. Lignifi ed integuments, however, were indistinct or absent from crystalliferous ray cells (Figures 2a-e).

Analysis of the crystals by X-ray diff raction indicated that calcium oxalate was present as silicates in the taxa of 3 diff erent sections. Th e FTIR spectrum revealed several absorption bands at 608-897, 1046-1739, and 2346-3436 cm−1. In Q. cerris L. var. cerris and Q. aucheri amorphous silica exhibited a relatively strong peak at 800 nm, separate from the band of crystalline silicate (15). Th e structure of most polymorphous SiO

2, both crystalline and

amorphous, is based on a tetrahedral unit of silicon coordinated to 4 oxygen atoms. Amorphous silica is one of the polymorphous forms of silica, and at high temperatures it can easily transform from quartz. In

the Si-O-Si bending vibration region (400-700 cm–1) of quartz, the band at 695 cm–1 determines whether it is crystalline or amorphous (13). In the amorphous state this band will be missing. Th e absorption band at 695 cm–1 is due to vibrations in octahedral site symmetry, at 780 cm–1 it is due to vibrations in tetrahedral site symmetry (16,17), and 781 cm–1 is present only in Q. macranthera. Tetrahedral symmetry is stronger than octahedral symmetry. Th erefore, in any structural change, the damage occurs fi rst in octahedral and then in tetrahedral symmetry. When the temperature was rapidly brought to 600-700 °C, the SiO

4 tetrahedral could not be ordered into a

crystalline state and was preserved in an amorphous silicate phase. Th e carbonate structure contains an isolated CO-2

3 group with a doubly degenerate

symmetric stretch (ν3) in the 1507-1599 cm–1 region


389

(18). Th e absorption bands at 3420, 3429, and 3436

cm–1 and 1624, 1633, and 1644 cm–1 arise due to the

adsorbed water molecules commonly observed in

natural silica (17). Th e XRF results show that the

major component of the sample is SiO2. Th e broad

Bragg peaks observed in X-ray diff raction studies

reveal that the sample could be nanocrystalline

quartz. Figure 3 shows the XRD spectra of CaOx

and CaOx-SiO2 composites formed in water. COM

was found to be the only crystalline phase present in

CaOx (Figure 3a), and the composite was obtained in

all 3 species of Quercus (Figures 3b, c) (19). Th e OH

stretching of coordinated COM water molecules was

demonstrated by broad absorption bands between

3000 and 3500 cm−1 in all samples. Th e water

calibration bands of the composites (660 and 584

cm−1) were shift ed slightly so that they were lower

than those of CaOx (667 and 594 cm−1). Th is was

a b c

e

d

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.


390

probably due to an increase in the number of water molecules in the crystal structure (i.e. the presence of COT) (20). Th is observation was in contrast to the results of XRD and SEM imaging in which COT was the main component of the composite formed only in Q. macranthera taxa. Due to the Si-O-Si stretching vibration of amorphous silica, another weak band was present at 1113 cm−1 in the CaOx-SiO

2 composite

deposit spectrum (Figure 3d) (21). Th e other 2 taxa, Q. aucherii and Q. cerris var. cerris, have 608 cm−1

bands of composite characteristics. SEM imaging of composite materials in Q. macranthera wood (Figures 3d-h) showed a mixture of hexagonal platelets of COM and needles of COT. Aggregates of monoclinic COM crystals, with contact twinning [attachment of 2 individual crystals along (1 0 0) plane] being more prevalent in the binary mixture (Figure 3d). FT-IR was used to analyse the molecular structure of sample components. Th e IR spectra of CaOx and composites exhibited peaks around 1628 cm−1, 1317 cm−1 (symmetrical and antisymmetrical oxalate C=O bond stretching), 781 cm−1 (O-C-O in-plane bending), and 517 cm−1 (O-C-O in-plane rocking), which is characteristic of COM (20).

Discussion

Th e presence of CaOx crystals as composites in the 3 diff erent Quercus taxa represents important phylogenetic, systematic, and evolutionary evidence. Th ese structures are formed as biominerals of diff erent morphologies (raphides, prisms, druses, and sand) and silicon dioxide bodies, which are taxon-specifi c and heritable (22). Silica accumulation was especially prevalent in Commelinids (Araceae, Commelinales, Poales, and Hanguana) and Orchids (23). In Quercus species, whewellite (Ca oxalate monohydrate) prismatic crystals were observed; only in Q. macranthera were weddellite (Ca oxalate trihydrate) crystals found together with composite whewellite forms. When we investigated taxonomically and phylogenetically, the coexistence of the needle-shaped trihydrate and the monohydrate forms of calcium oxalate inside the same plant cell demonstrated synapomorphy among the Quercus taxa; however, the occurrence of silicates as calcium oxalate composites show symplasty between the monocots and Quercus taxa. Hydrated forms

of calcium oxalate occur naturally as 3 mineral species: whewellite (monohydrate, known from some coal beds), weddellite (dihydrate), and a very rare trihydrate called caoxite (24). Macrocrystals of the polyhydrate were found in the fossil deposits of the Weddell Sea and named weddellite. Its crystal structure was disclosed in a kidney calculus and is isomorphic with tetragonal strontium oxalate.

Th e evolutionary background of silicifi cation demonstrates that diatoms, which have molecular genetic similarities among the silicic acid transporters of these organisms (ochristian algae), were known to be the single origin of silicifi cation; however, there is no sequence similarity among the 2 silicic acid transporters known from Oryza sativa and those known from ochristian algae (25-27) or the putative transporter from sponge (28). Among the vascular plants, Poales and Equisetales have a shared hemicellulose component that could be a template for silica deposition, although they are phylogenetically distant within the euphyllophyte plants (29,30). For terrestrial plants there is no signifi cant fossil evidence on which to base the environment-evolution interactions in the production of alkaline earth biominerals such as calcium oxalate, despite both silica and calcium carbonate deposition in the ocean throughout the Phanerozoic. However, the impact of mineralising photosynthetic organisms on the availability of soluble substrates for mineralisation for calcifi ers has been smaller than the impact for silifi ers (31). Lauraceae and Fagaceae, tropical plants that have fossils from the Cretaceous, provide an example of the eff ect of environmental factors (32). Th e wood anatomy of some Lauraceae from the Amazonian forest and the Atlantic forest of south-eastern Brazil demonstrate the occurrence of silica bodies in ray cells (33). Among the diff erent Quercus taxa, Q. macranthera must be later than the other Quercus taxa phylogenetically because it contains whewellite and Ca oxide (Ca oxalate trihydrate) together inside the plant. Elsewhere in the plant kingdom a few observations have been reported. Opuntia fi cus-indica for example, has highly productive crassulacean acid metabolism (CAM). In the parenchymal tissues of Opuntia fi cus-indica (Miller) 2 forms of calcium oxalate that diff er in hydration level were identifi ed (34). In a study conducted with sessile oak wood, crystals were


391

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).

fe

hg


392

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Acknowledgements

We want to express our special thanks to Dr. Pieter Baas for his valuable guidance and critical reading of the manuscript. A condensed version of this article was presented at the First International Symposium on Turkish-Japanese Environment and Forestry (4-6 November 2010).

Corresponding author:

Bedri SERDAR

Karadeniz Technical University,

Faculty of Forestry,

61080 Trabzon-TURKEY

Email: [email protected]


393

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Calcium oxalate crystal types in three oak species