Characterization of Calcium Oxalate Biominerals in Pereskia Species (Cactaceae) Paula V

Characterization of Calcium Oxalate Biominerals in Pereskia Species (Cactaceae) Paula V. Monje and Enrique J. Baran* Centro de Química Inorgánica (CEQUINOR, CONICET/UNLP), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, C. Correo 962, 1900 La Plata, Argentina. E-mail: [email protected] * Author for correspondence and reprint requests Z. Naturforsch. 64 c, 763 – 766 (2009); received June 29, 2009 Calcium oxalate druses were isolated from the stems and leaves of six Pereskioideae family members and investigated by infrared spectroscopy, showing that in all samples the biomineral was present in the form of whewellite, CaC2O4 · H2O. As Pereskia is thought to represent the “ancestral” condition of the leaﬂ ess stem-succulent cacti, these results suggest that the biomineralization of calcium oxalate in Cactaceae represents a primitive characteristic of the group and also support a close genetic relationship between Pereskia and Opuntia. Key words: Pereskia, Biominerals, Whewellite, Infrared Spectroscopy

Introduction excretory systems to eliminate such excess, they modulate the difference between the natural Crystalline oxalates are widely distributed in abundance of calcium and the very low intra- nature and have been observed in rocks, soil, and cellular levels required by tightly controlling the among a variety of living organisms, including distribution of calcium and its compartmentation plants and animals (Baran and Monje, 2008). The within cells. In this context, with a solubility prod- most widely distributed biomineral of this type uct of 1.3 · 10 – 9 mol2 l–2 in water, calcium oxalate is calcium oxalate, which is especially common effectively sequesters calcium and renders itself in the plant kingdom (Arnott, 1982; Khan, 1995; metabolically and osmotically inactive for plant Monje and Baran, 2004a; Baran and Monje, 2008). cells. Therefore, many plants accumulate crystal- The presence of other metal oxalates is extremely line calcium oxalate in response to the presence rare in biological systems, although the presence of an excess of calcium (Webb, 1999; Baran and of crystalline magnesium, manganese, copper, and Monje, 2008). ammonium oxalates in different forms of life has been reported, whereas soluble forms of sodium On the other hand, a number of recent studies and potassium oxalates are widely distributed in indicate that these calcium oxalate crystals do not plants and fungi (Baran and Monje, 2008). form an inert, non-retrievable deposit of calcium, Crystalline calcium oxalates have been found in but can act as a sort of reservoir to which plants two different hydration states in plants, either as can recur in cases of calcium defi ciency. Apart from this primary function as a calcium regula- the monoclinic monohydrate (whewellite, CaC2O4 tor, the great variety of crystal shapes and sizes, · H2O) or as the orthorhombic dihydrate (wed- as well as their localization in different plant tis- dellite, CaC2O4 · 2H2O). Whewellite is the most stable form from the thermodynamic point of sues, suggests various other functions for calcium view (Monje and Baran, 2004a; Baran and Monje, oxalates which might be evolved secondarily. 2008). However, the morphology (druses, raphids, Some of these functions include mechanical sup- solitary crystals, crystal sand) and the hydration port, gravity perception, intracellular pH regula- state of calcium oxalates in plants differ signifi - tion and ion balance, detoxifi cation and even light cantly among different groups. gathering and refl ection (Nakata, 2003; Baran and Because higher plants incorporate calcium in Monje, 2008). excess to the cellular requirements and most of The Cactaceae family is a well-known lineage them, unlike animals, do not have well-developed of stem-succulent plants that are widely recog-

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nized for their specialized adaptation to drought. healthy and well-hydrated specimens growing un- These include an extensive and shallow root sys- der normal greenhouse conditions, regardless of tem with dynamic hydraulic properties that al- the season and other environmental conditions. low for maximum water uptake following rain, a succulent pith and cortex with high capacitance Crystal isolation and purifi cation for water storage, and a long-lived stem corti- Crystal druses were isolated from fresh plant cal tissue system that has replaced leaves as the tissues. Plant stems and leaves were carefully primary photosynthetic organ. These anatomi- washed with abundant distilled water to remove cal and histological adaptations come along with any possible external contamination, cut with the acquisition of crassulacean acid metabolism a razor blade into thin tissue sections and im- (CAM photosynthesis), which minimizes the tran- mersed in a 6% sodium hypochlorite solution for spirational water loss by allowing the stomata to 48 h. The tissue was then mechanically dissociated open during the night (Edwards and Diaz, 2006; by passing it repeatedly through a plastic trans- Edwards and Donoghue, 2006). Cactaceae species fer pipette, fi ltered through gauze, and allowed are also unique in their ability to biomineralize to decant for 30 min. The pellet containing crys- calcium oxalate (Rivera and Smith, 1979; Monje talline products was suspended in absolute etha- and Baran, 2002; Hartl et al., 2007), and the con- nol, and the crystals were manually collected by tent of the biomineral can reach up to 80 – 90% of inspection through a dissecting light microscope. the plant’s dry weight (Monje and Baran, 2004a). The isolated crystalline material was repeatedly However, the connection between calcium oxalate washed with ethanol until the tissue debris was no biomineralization and water stress adaptation re- longer evident. This procedure renders crystalline mains obscure. samples consisting mainly of intact druses. The The Pereskia genus (subfamily: Pereskioideae, purifi ed crystal samples were fi nally dried under family: Cactaceae) consists of species of leafy a nitrogen fl ow before spectroscopic analysis. shrubs and trees and is the only cactus group that has persistent non-succulent stems and leaves. It Synthesis of CaC2O4 · H2O samples has long been considered as the best living rep- resentation of the “ancestral cactus” (Edwards et For comparative purposes we have prepared al., 2005; Edwards and Donoghue, 2006; Metzing high-purity crystalline CaC2O4 · H2O samples. and Kiesling, 2008). As opposed to succulent cac- They were prepared by simultaneous dropping of ti, most species of Pereskioideae grow in tropical, highly diluted sodium oxalate and calcium chlo- humid environments. ride solutions in a great volume of distilled water, We have extensively studied the biominerals maintained at 70 °C, as described by Grases et al. present in the two main succulent subfamilies of (1990). cacti, Cereoideae and Opuntioideae, and have found the presence of calcium oxalate (Monje Infrared spectroscopy and Baran, 1996, 1997, 2002, 2004b), magnesium The IR spectra were obtained by means of a oxalate (Monje and Baran, 2005), and silicon di- Bruker IFS 66 spectrophotometer in the spectral oxide (Monje and Baran, 2000, 2004b). We have range between 4000 and 400 cm–1 using the KBr now investigated the presence of calcium oxalates pellet technique (4 mg of the powdered sample in different species of Pereskia, the non-succulent dispersed in 100 mg of KBr). “relictual cacti”, in order to investigate the possible relationship between calcium oxalate biomin- Results and Discussion eralization and adaptation of cacti drought. Infrared spectroscopy is a powerful tool for the Material and Methods investigation of the chemical composition of plant material (Baran, 2005); we have applied this meth- Plant material odology successfully in the investigation of plant The plant material was provided by the South biominerals (Monje and Baran, 1996, 1997, 2000, Florida Cactus and Succulent Society (Miami, 2002, 2004b, 2005; Baran and Rolleri, 2009). In the Florida). The stems and leaves of six representa- present study, we have used this sensitive spectro- tive species of Pereskia were harvested from scopic technique for the fi rst time to analyze the

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composition of crystalline material isolated from ellite is the main biomineral present in the druses the stems and leaves of six different members of of Pereskioideae members. Four other species of the Pereskiodeae subfamily (Pereskia aculeata, P. this family (P. grandifolia, P. sacharosa, P. stenan- bleo, P. portulacifolia, P. nicoyana, P. quisqueyana, tha, P. zinniﬂ ora) recently, investigated by X-ray and Pereskiopsis sp.); all species showed the pres- diffractometry, also show the presence of whewel- ence of highly abundant druses in tissues of the lite (Hartl et al., 2007). stems and leaves. Our previous investigations on biominerals The infrared spectra of biominerals present in present in the Cactaceae family have shown that the puriﬁ ed druses of Pereskia species showed Opuntioideae family members biomineralize cal-

clearly that the crystals consisted of highly pure cium oxalate as whewellite, CaC2O4 · H2O, where- whewellite (CaC2O4 · H2O). This was a consist- as Cereoideae members biomineralize calcium

ent ﬁ nding regardless of the species analyzed and oxalate as weddellite, CaC2O4 · 2H2O, suggesting their localization in the plant body. Fig. 1 shows a deﬁ nite but different genetic control over the a comparison of the infrared spectra of a pure biomineralization process in the two most rep-

synthetic sample of CaC2O4 · H2O a representa- resentative groups of the succulent Cactaceae tive crystalline sample of Pereskia (P. bleo), and a (Monje and Baran 2002, 2004a). This conclusion crystalline sample isolated from an Opuntioideae was confi rmed by a recent X-ray diffraction study family member (Tephrocactus articulatus), that of the total crystalline material present in a large was identifi ed as whewellite in a previous study number of Cactaceae species (Hartl et al., 2007). (Monje and Baran, 2002). As it can be seen the Even though the simultaneous presence of the three spectra are identical, indicating that whew- two hydration states of calcium oxalate in a single taxon seems to be rare in angiosperms (Arnott, 1982), Hartl et al. (2007) showed that certain Cac- taceae species display the concurrent presence of weddellite and whewellite. One of the few known examples in which the presence of both forms of calcium oxalate has been unambiguously demon- strated is Opuntia fi cus indica (Malainine et al., 2003). Overall, the results of our study confi rmed a close genetic relationship between Pereskia and Opuntia (Metzing and Kiesling, 2008), as both groups biomineralize calcium oxalate as whewellite druses, which were also highly similar in size and morphology. Therefore, we conclude that whewellite must have been the ancestral biomineral in the Cactaceae lineage and that weddellite must have evolved after branching of the Cereus as a separate group. Besides, the observation of whewellite druses in all species of Pereskia suggests that the biomineralization of calcium oxalate in Cactaceae represents a primitive characteristic of the family, most likely acquired before adaptation to water stress conditions. Whereas primitive cacti deposit calcium oxalate in the form of the thermodynamically most stable monohydrate, in the course of evolution certain lines of Cactace- ae perhaps conserved calcium oxalate mainly in Fig. 1. FTIR-spectra of (A) a pure sample of synthetic the form of the metastable and convertible dihy- CaC2O4 · H2O, (B) druses of Pereskia bleo and (C) druses of Tephrocactus articulatus. drate.

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Acknowledgements la República Argentina (CONICET). E. J. B. is This work was supported by Universidad Na- a member of the Research Career of this organ- cional de La Plata (UNLP) and Consejo Nacion- ism. al de Investigaciones Cientíﬁ cas y Técnicas de

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