Isolation of a Crystal Matrix Protein Associated with Calcium Oxalate Precipitation in Vacuoles of Specialized Cells

Abstract

The formation of calcium (Ca) oxalate crystals is considered to be a high-capacity mechanism for regulating Ca in many plants. Ca oxalate precipitation is not a stochastic process, suggesting the involvement of specific biochemical and cellular mechanisms. Microautoradiography of water lettuce (Pistia stratiotes) tissue exposed to 3 H-glutamate showed incorporation into developing crystals, indicating potential acidic proteins associated with the crystals. Dissolution of crystals leaves behind a crystal-shaped matrix "ghost" that is capable of precipitation of Ca oxalate in the original crystal morphology. To assess whether this matrix has a protein component, purified crystals were isolated and analyzed for internal protein.Polyacrylamide gel electrophoresis revealed the presence of one major polypeptide of about 55 kD and two minor species of 60 and 63 kD. Amino acid analysis indicates the matrix protein is relatively high in acidic amino acids, a feature consistent with its solubility in formic acid but not at neutral pH.45 Ca-binding assays demonstrated the matrix protein has a strong affinity for Ca. Immunocytochemical localization using antibody raised to the isolated protein showed that the matrix protein is specific to crystal-forming cells. Within the vacuole, the surface and internal structures of two morphologically distinct Ca oxalate crystals, raphide and druse, were labeled by the antimatrix protein serum, as were the surfaces of isolated crystals. These results demonstrate that a specific Ca-binding protein exists as an integral component of Ca oxalate crystals, which holds important implications with respect to regulation of crystal formation.Many plants produce calcium (Ca) oxalate as crystalline deposits (Arnott and Pautard, 1970; Gallaher, 1975; Franceschi and Horner, 1980; Horner and Wagner, 1995; Webb, 1999; Nakata, 2003), which can account for greater than 85% of the dry weight of some plant organs. The formation of Ca oxalate is an essential process in many species, and more than 90% of tissue Ca can be tied up as this compound (Gallaher et al., 1975; Gallaher and Jones, 1976). Ca oxalate crystals often occur within the vacuole of crystal idioblasts (Foster, 1956), specialized cells that generally encompass less than 1% to 2% of the total cells of the Ca-accumulating tissue. Because Ca oxalate formation is the end result of a mechanism for controlling Ca at the tissue and organ levels in the plant (Zindler-Frank, 1975; Borchert, 1985; Franceschi, 1989 Franceschi, , 2001 DeSilva et al., 1996; Kuo-Huang and Zindler-Frank, 1998; Pennisi and McConnell, 2001; Zindler-Frank et al., 2001; Volk et al., 2002), cells producing the crystals are considered to be highcapacity Ca sinks. Because crystal idioblasts perform a unique complex function of importance to the general physiology of the plant, and they commonly occur as single cells scattered among other tissues, we have referred to them as single-celled organs (Kostman and Franceschi, 2000).Large amounts of Ca oxalate crystals can be formed...

Mass spectroscopic characteristics of low molecular weight proteins extracted from calcium oxalate stones: preliminary study

“…In addition, not all amino acids in a mass of proteins exert a specialized function in biological fields. Furthermore, acidic amino acids were reported to be found within stones and it was hypothesized that they are involved in the formation of stones in specialized plant cells (2). Understanding the characteristics of amino acids in peptide or proteins might help in the understanding of how proteins are related to stone formation.…”

“…It has been reported that proteins are involved in the formation of calcium oxalate depositions in plants (2). Therefore, proteins that present in the stones or urine can be considered to modify crystal formation.…”

Structural Changes and Fate of Crystalloplastids during Growth of Calcium Oxalate Crystal Idioblasts in Japanese Yam (Dioscorea japonica Thunb.) Tubers

“…Despite many electron microscopic studies showing the crystal chambers and crystal-associated materials in the vacuoles of crystal idioblasts, little was known about their origin. Recently, Li et al (2003) reported that the matrix protein in calcium oxalate crystals was transported to the vacuoles via the Golgi apparatus. In Japanese yam, there were amorphous materials in the translucent parts of crystalloplastids.…”

The polymorphic weddellite crystals in three species of Cephalocereus (Cactaceae)

“…Moreover, the diffraction pattern revealed that in the species studied there was only one mineral, 90% calcium as shown by EDXAR analysis, being the first report of the elemental composition of the core from aggregated crystals of weddellite. In vacuoles, which are environments rich in water and ionic solutions, the signal could be provided when different factors are present, such as a decrease in the oxalate-oxidase enzyme (Volk et al, 2002), an increase in the glycolate concentration (Yu et al, 2010), or the formation of a protein matrix (Li et al, 2003), as occurs in the other plants (Horner and Wagner, 1995). Some of these factors may contribute to the quick weddellite precipitation in Cephalocereus.…”