A highlightedly improved method for isolating and characterizing calcium oxalate crystals from tubercles of Mammillaria schumannii.

BACKGROUND: Calcium oxalate (CaOx) is the most prevalent and widespread biomineral in plants and is involved in protective and/or defensive functions against abiotic stress factors. It is, however, expected that this function has an extremely significant contribution to growth processes in plants bearing large amounts of CaOx, such as cacti growing in desert environment. RESULTS: In our research, small-sized CaOx crystals (≤ 20 µm) with tetrahedral or spherical shapes were observed to dominate in each epidermal and cortical cell from the tubercles of Mammillaria schumannii, a species from the Cereoideae subfamily, having tubercles (main photosynthetic organs) united with adjacent ones almost into ridges on its stem. Because they have potential significant functions, differential centrifugations after mechanical blending were used to obtain these small-sized CaOx crystals, which extremely tend to adhere to tissue or suspend in solution. And then the combined Scanning Electron Microscope Energy Dispersive System (SEM-EDS) and Raman spectroscopy were further performed to demonstrate that the extracted crystals were mainly CaC2O4·2H2O. Interestingly, spherical druses had 2 obvious abnormal Raman spectroscopy peaks of -CH and -OH at 2947 and 3290 cm-1, respectively, which may be attributed to the occluded organic matrix. The organic matrix was further extracted from spherical crystals, which could be polysaccharide, flavone, or lipid compounds on the basis of Raman spectroscopy bands at 2650, 2720, 2770, and 2958 cm-1. CONCLUSIONS: Here we used a highlightedly improved method to effectively isolate small-sized CaOx crystals dominating in the epidermal and cortical cells from tubercles of Mammillaria schumannii, which extremely tended to adhere plant tissues or suspend in isolation solution. And then we further clarified the organic matrix getting involved in the formation of CaOx crystals. This improved method for isolating and characterizing biomineral crystals can be helpful to understand how CaOx crystals in cacti function against harsh environments such as strong light, high and cold temperature, and aridity.