Characterization of Calcium Oxalate Biominerals in Some (Non-Cactaceae) Succulent Plant Species 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. 65 c, 429 – 432 (2010); received February 5, 2010 The water-accumulating leaves of crassulacean acid metabolism plants belonging to fi ve different families were investigated for the presence of biominerals by infrared spectro- scopic and microscopic analyses. Spectroscopic results revealed that the mineral present in succulent species of Agavaceae, Aizoaceae, and Asphodelaceae was calcium oxalate mono- hydrate (whewellite, CaC2O4 · H2O). Crystals were predominantly found as raphides or soli- tary crystals of various morphologies. However, representative Crassulaceae members and a succulent species of Asteraceae did not show the presence of biominerals. Overall, these results suggest no correlation between calcium oxalate generation and crassulacean acid metabolism in succulent plants. Key words: Succulent Plants, Biominerals, Whewellite, Crassulacean Acid Metabolism Introduction With the exception of some Pereskioideae species, all Cactaceae members are succulents, a The succulent photosynthetic stem parenchyma diverse group of plants that store water in their of Cactaceae family members contains abundant leaves, stems and/or roots as an adaptive trait to calcium oxalate crystals. Whereas Opuntioideae arid soils, high temperature, and prolonged water (pad-like cacti) and Pereskioideae (ancestral cac- defi cit. Most succulent plants also display other ti) species preferentially deposit calcium oxalate water-saving features that include: 1) a reduced monohydrate (CaC2O4 · H2O, whewellite), Cereo- outer surface that confers the plants a typical co- ideae species (columnar cacti) show predomi- lumnar or spherical shape; 2) reduced or absent nance of calcium oxalate dihydrate (CaC2O4 · leaves and a stem adapted to perform photosyn- 2H2O, weddellite). In Cactaceae, crystals of cal- thesis; 3) a waxy, hairy or multi-layered epider- cium oxalate are found as druses, i.e. multi-crystal mis with a reduced number of stomata, which are agglomerates with an overall spherical shape, re- closed during the day and open during the night; gardless their chemical composition (Monje and 4) a type of photosynthesis known as crassu- Baran, 2002). Interestingly, the biomineralization lacean acid metabolism (CAM), which allows of calcium oxalate in Cactaceae shows some ex- the fi xation of carbon dioxide during the night clusive features: 1) the content of calcium oxalate thereby minimizing the loss of water through the in photosynthetic tissues can reach up to 80 – 90% open stomata (Bowyer and Leegwood, 1997). The of the plants’ dry weight; 2) most Cereoideae relationship between CAM and calcium oxalate members show large diameter druses of highly formation is at present unclear. pure and stable weddellite, a thermodynamically In this study, we investigated the presence of metastable form of calcium oxalate which is rare- bio minerals in CAM species not related to Cacta- ly found in living organisms under the form of a ceae. Abundant crystals with different morpholo- druse because it easily converts into whewellite; gies were isolated from the succulent photosyn- and 3) some species of cacti have shown co-exist- thetic tissues of all analysed succulent species of ence of whewellite and weddellite in a single or- Agavaceae, Aizoaceae, and Asphodelaceae. Con- ganism (Baran and Monje, 2008; Hartl et al., 2007; trary to Cactaceae, the isolated crystalline materi- Malainine et al., 2003). al was predominantly found in the form of raphi- 0939 – 5075/2010/0700 – 0429 $ 06.00 © 2010 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D 430 P. V. Monje and E. J. Baran · Biominerals in Succulent Plants des, i.e. bundles of elongated needle-like crystals, with ethanol until tissue debris was no longer evi- and the spectroscopic analysis revealed that the dent. This procedure rendered crystalline samples biomineral present was always whewellite. consisting mainly of intact raphides and various quantities of solitary crystals of different mor- Material and Methods phologies, i.e. styloids and prismatic crystals. Plant material Infrared spectroscopy The succulent leaves of a number of represen- Purifi ed samples were dried under a nitrogen tative CAM species were harvested from healthy fl ow before spectroscopic analysis. The IR spec- and well-hydrated specimens growing under tra were obtained by means of a Bruker IFS 66 normal greenhouse conditions, regardless of the spectrophotometer in the spectral range between season and other environmental conditions. The 4000 and 400 cm–1 using the KBr pellet tech- plant material was kindly provided by the South nique (4 mg of the powdered sample dispersed in Florida Cactus and Succulent Society (Miami, FL, 100 mg of KBr). The samples were determined as USA). whewellite, by comparison with results from pre- vious studies (Monje and Baran, 2002, 2009). Crystal isolation and purifi cation The crystalline material was isolated from fresh Phase contrast microscopy plant tissues following a previously described For microscopic observations, freshly isolated method (Monje and Baran, 2009). Briefl y, the crystals were suspended in water and mounted plant tissue was cut into thin slices using a razor on glass slides for phase contrast microscopy on blade and immersed in 6% sodium hypochlorite an Olympus IX-70 inverted microscope, using solution for 48 h. The digested tissue sections were 10 – 40× dry objectives. Black and white digital disrupted mechanically and fi ltered through ster- pictures were processed and arranged for pres- ile gauze. The fi ltered extract was allowed to de- entation using Adobe Photoshop 7.0 and Adobe cant for at least 30 min in a test tube or collected Illustrator CS3. by low-speed centrifugation (1,000 rpm, 10 min). The resulting pellet containing crystalline prod- Results and Discussion ucts was suspended in absolute ethanol, and the crystals were collected by inspection through a In this study, we performed an infrared spec- stereomicroscope. Crystals were repeatedly rinsed troscopic and microscopic analysis of the biomin- Fig. 1. Representative crystal shapes of calcium oxalate (CaC2O4 · H2O, whew- ellite) from non-Cactaceae succulent species: (a, b) Gasteria liliputana; (c) Aloe nobilis; (d, e) A. harliana, and (f, g) Faucaria stomatium. Intact raphides still enclosed within the cell walls of the crystal-forming cells (a, c, and f) were typically found in the isolated preparations. Detailed images of the individual crystals that compose the raphides are shown in b, e, and g. Oth- er crystal morphologies, e.g. styloids (d), and prismatic crystals (c and d, arrowheads) were less abundant but usually present in most of the ana- lysed samples. Scale bars, 10 μm. P. V. Monje and E. J. Baran · Biominerals in Succulent Plants 431 Table I. Microscopic and IR-spectroscopic analysis of biominerals isolated from the leaves of non-Cactaceae suc- culent plants species. Family Genus Crystalsa Morphology Chemical compositionb Agavaceae Agave parrasana Yes, higly abundant Raphides (abundant) Whewellite Styloids (few) Prismatic crystals (few) Agave victoria Yes, abundant Raphides (abundant) Whewellite Styloids (few) Aizoaceae Faucaria stomatium Yes, abundant Raphids (abundant) Whewellite Prismatic crystals (few) Asphodelaceae Aloe nobilis Yes, abundant Raphides (abundant) Whewellite Prismatic crystals (few) Aloe harlana Yes, abundant Raphides (abundant) Whewellite Gasteria liliputana Yes, abundant Raphides (abundant) Whewellite Haworthia obtusa Yes, abundant Raphides (abundant) Whewellite Asteraceae Senecio citriformis Not observed Crassulaceae Sedum pachyphyllum Not observed Graptopetalum Not observed amethystimum Adromischus maculatus Not observed a Microscopic observations. b Determined by IR spectroscopy. erals present in the leaves of water-accumulating species of Crassulaceae and a succulent species of species of Agavaceae, Aizoaceae, Asphodelaceae, Asteraceae. The absence of crystals in these spe- Crassulaceae, and Asteraceae (formerly named as cies was confi rmed by microscopic observations Compositae). Infrared spectroscopy is a powerful of live tissue sections. tool for the investigation of the chemical com- Despite the widespread occurrence of calcium position of plant material (Baran, 2005) and we oxalates in the plant kingdom, limited informa- have used this methodology successfully in earlier tion has been given on the presence of calcium investigations of plant biominerals, including cal- oxalates in succulent species displaying CAM, cium oxalates (e.g. Monje and Baran, 2002, 2009; with the exception of Cactaceae (Rivera and Baran and Rolleri, 2009), magnesium oxalates Smith, 1979; Monje and Baran, 2002; Hartl et al., (Monje and Baran, 2005) and silicon dioxide (e.g. 2007; Baran and Monje, 2008). The results from Monje and Baran, 2000). the present study indicate that calcium oxalates The results from the present study are summa- are also abundant in the succulent tissues of Aga- rized in Table I. Fig. 1 shows representative crys- vaceae, Asphodelaceae, and Aizoaceae species. tal shapes found in the isolated material of some Consistent with our fi ndings, two species of Aga- of the investigated