Isolation of a crystal matrix protein associated with calcium oxalate precipitation in vacuoles of specialized cells

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 3H-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. 45Ca-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.     

The use of a laser microprobe to identify calcium oxalate in histological material

Summary Some birefringent renal deposits were found to give the usual histological reactions for calcium oxalate but were soluble in caustic alkalis. Comparison with known oxalate crystals using a laser microprobe mass analyser confirmed the presence of calcium oxalate. Similar crystals were found in liver tissue from a rat poisoned with ethylene glycol.     

CALCIUM OXALATE CRYSTALS IN THE GREEN ALGA SPIROGYRA SP. (ZYGNEMATALES, CHLOROPHYTA)

Specimens of an unidentified species of the freshwater green alga Spirogyra were found to have abundant cruciate cellular inclusions up to 34 micrometers long. A crystalline nature was shown by birefringence in polarized light. Despite their large size and complex shape, these inclusions did not occur free in the large central vacuole. Instead, they were associated with cytoplasmic strands that spanned the space between gyres of the parietal spiral chloroplasts and with strands that suspended the nucleus in a cytoplasmic embayment of the central vacuole. Some crystals moved directionally along the cytoplasmic strands, and their movement was arrested by cytochalasin B, suggesting that actin microfilaments had a role in crystal movement. Solubility tests showed that the inclusions were composed of calcium oxalate; they dissolved rapidly in weak hydrochloric acid without effervescence, but they were not soluble in concentrated acetic acid or sodium hypochlorite. A colorimetric enzymatic test for oxalate was used to demonstrate microscopically the presence of oxalate and to quantify the amounts. The calcium oxalate crystals were surrounded by a water-soluble organic matrix that retained the shape of the crystal even after demineralization. Scanning electron microscopy was used to examine the morphology of isolated crystals.     

A calcium oxalate-secreting tissue in branchlets of the Casuarinaceae

Summary Deciduous branchlets of casuarina trees have an unusual calcium oxalate-secreting system in which the epidermal tissue deposits calcium oxalate crystals in cell walls of the branchlet surface. These prismatic crystals were identified by light and electron microscopy, histochemistry, and elemental X-ray analysis. This calcium oxalate-secreting tissue was found in all species of casuarinas examined, including three of the four genera of the Casuarinaceae:Allocasuarina sp.,Casuarina sp., andGymnostoma papuanum. Because crystals were present throughout the epidermis soon after it formed, the mechanism for their induction was likely to be different than that for calcium oxalate crystal idioblasts. Secreting cells had a complex endoplasmic reticulum that may be involved in the secretory process.Abbreviations EDS energy-dispersive X-ray spectroscopy - HPF/FS high pressure-frozen/freeze-substituted - SEM scanning electron microscopy - TEM transmission electron microscopy Dedicated to the memory of Professor John G. Torrey     

Cytoplasmic streaming of calcium oxalate crystals in Callipsygma wilsonis (Bryopsidales, Chlorophyta)

Light microscopic study of the giant‐celled, marine green alga Callipsygma wilsonis J. Agardh (Udoteaceae, Bryopsidales) revealed numerous birefringent crystalline inclusions in the terminal segments of the assimilatory axes. The inclusions were thin plates with a triangular shape in face view, a base up to 75 μm in length, and a height that was one‐seventh the length of the base. Crystals of various sizes commonly were stacked face‐to‐face with one or more edges coinciding, but removal of organic material by treatment in sodium hypochlorite resulted in disaggregation. The crystals were soluble in dilute hydrochloric acid without effervescence but were insoluble in acetic acid. These diagnostic chemical solubility tests and a positive reaction to the Yasue staining reaction indicated that the crystals were composed of calcium oxalate. Scanning electron microscopy showed that most crystals had smoothly curving edges, but some had truncate or beveled margins. Calcium oxalate crystals have been reported to occur in the large central vacuoles of several bryopsidalean species, but the crystals in C. wilsonis were present in the parietal cytoplasm, which was evident from the presence of crystals in streaming cytoplasm. Calcium oxalate crystals, amyloplasts, chloroplasts, and other cytoplasmic constituents moved along cytoskeletal cables at rates of approximately 2.8 μm s⁻¹. These findings add to a growing body of evidence that calcium oxalate crystals in diverse algae may be present in cellular compartments other than the central vacuole.     

Localization of ATP-dependent calcium transport activity in mouse pancreatic microsomes

Electron-dense deposits representing calcium oxalate crystals which result from ATP-dependent calcium uptake have been localized within vesicles of of a heavy microsomal fraction prepared from mouse pancreatic acini. In the absence of either ATP or oxalate, no electron-dense deposits could be observed. By subfractionation of microsomes on discontinuous sucrose gradients, it could be shown that the highest energy-dependent calcium transport activity was associated with the rough endoplasmic reticulum. In rough microsomes, the 45Ca2+-uptake measured was 7 times greater than that of smooth microsomes in the presence of ATP and oxalate and about 3 times greater in he presence of ATP alone. When ribosomes were released from the rough endoplasmic reticulum vesicles by treatment with KCl in the presence of puromycin, the stripped microsomes showed a 40% increase in the specific 45Ca2+-uptake activity measured in he presence of ATP and oxalate and an increase of 80 to 90% in the presence of ATP alone. From these results it can be concluded that the calcium transport activity of microsomes prepared from mouse pancreatic acini is located predominantly in the rough endoplasmic reticulum membrane.     

Calcium oxalate crystals in plants

Calcium (Ca) oxalate crystals occur in many plant species and in most organs and tissues. They generally form within cells although extracellular crystals have been reported. The crystal cells or idioblasts display ultrastructural modifications which are related to crystal precipitation. Crystal formation is usually associated with membranes, chambers, or inclusions found within the cell vacuole(s). Tubules, modified plastids and enlarged nuclei also have been reported in crystal idioblasts. The Ca oxalate crystals consist of either the monohydrate whewellite form, or the dihydrate weddellite form. A number of techniques exist for the identification of calcium oxalate. X-ray diffraction, Raman microprobe analysis and infrared spectroscopy are the most accurate. Many plant crystals assumed to be Ca oxalate have never been positively identified as such. In some instances, crystals have been classified as whewellite or weddellite solely on the basis of their shape. Certain evidence indicates that crystal shape may be independent of hydration form of Ca oxalate and that the vacuole crystal chamber membranes may act to mold crystal shape; however, the actual mechanism controlling shape is unknown. Oxalic acid is formed via several major pathways. In plants, glycolate can be converted to oxalic acid. The oxidation occurs in two steps with glyoxylic acid as an intermediate and glycolic acid oxidase as the enzyme. Glyoxylic acid may be derived from enzymatic cleavage of isocitric acid. Oxaloacetate also can be split to form oxalate and acetate. Another significant precursor of oxalate in plants is L-ascorbic acid. The intermediate steps in the conversion of L-ascorbic acid to oxalate are not well defined. Oxalic acid formation in animals occurs by similar pathways and Ca oxalate crystals may be produced under certain conditions. Various functions have been attributed to plant crystal idioblasts and crystals. There is evidence that oxalate synthesis is related to ionic balance. Plant crystals thus may be a manifestation of an effort to maintain an ionic equilibrium. In many plants oxalate is metabolized very slowly or not at all and is considered to be an end product of metabolism. Plant crystal idioblasts may function as a means of removing the oxalate which may otherwise accumulate in toxic quantities. Idioblast formation is dependent on the availability of both Ca and oxalate. Under Ca stress conditions, however, crystals may be reabsorbed indicating a storage function for the idioblasts for Ca. In addition, it has been suggested that the crystals serve purely as structural supports or as a protective device against foraging animals. The purpose of this review is to present an overview of plant crystal idioblasts and Ca oxalate crystals and to include the most recent literature.     

Mercurous nitrate as a histochemical reagent for calcium phosphate in bone and pathological calcification and for calcium oxalate

Synopsis An aqueous solution of mercurous nitrate reacts with bone and tissue calcified sites with the formation of brown to black amorphous masses and feathery crystals, the last resembling the crystals formed from the action of an aqueous solution of mercurous nitrate on calcium orthophosphate. Calcium oxalate reacts with this mercurous nitrate solution to form brown to black deposits on the surface of the oxalate particles; this suggests an adsorption phenomenon. The brown deposits are blackened by ammonium hydroxide, gold chloride, and many sulphur-containing compounds.     

Isolated Medicago truncatula mutants with increased calcium oxalate crystal accumulation have decreased ascorbic acid levels.

The mechanisms controlling oxalate biosynthesis and calcium oxalate formation in plants remain largely unknown. As an initial step toward gaining insight into these regulatory mechanisms we initiated a mutant screen to identify plants that over-accumulate crystals of calcium oxalate. Four new mutants were identified, from an ethyl methanesulfonate (EMS)-mutagenized Medicago truncatula (cv. Jemalong genotype A17) population, that over-accumulated calcium oxalate crystals. The increased calcium oxalate content of these new mutants, as with the previously isolated mutant cod4, resulted from an increase in druse crystals accumulated within the mesophyll cells of leaves. Complementation and segregation analysis revealed that each mutant was affected at a different locus. This was confirmed through the genetic mapping of each mutation to different linkage groups. Together, these findings emphasize the complexity of factors that can contribute to oxalate biosynthesis and crystal formation in these plants. In addition, each mutant showed a common decrease in ascorbic acid content providing genetic support for ascorbic acid as a precursor in the oxalate biosynthetic pathway for druse crystal formation. Further support was obtained by the ability of an exogenous supply of ascorbate to induce druse crystal formation while other tested organic acids did not induce crystal production.     

Characterization of calcium oxalates generated as biominerals in cacti

The chemical composition and morphology of solid material isolated from various Cactaceae species have been analyzed. All of the tested specimens deposited high-purity calcium oxalate crystals in their succulent modified stems. These deposits occurred most frequently as round-shaped druses that sometimes coexist with abundant crystal sand in the tissue. The biominerals were identified either as CaC(2)O(4).2H(2)O (weddellite) or as CaC(2)O(4).H(2)O (whewellite). Seven different species from the Opuntioideae subfamily showed the presence of whewellite, and an equal number of species from the Cereoideae subfamily showed the deposition of weddellite. The chemical nature of these deposits was assessed by infrared spectroscopy. The crystal morphology of the crystals was visualized by both conventional light and scanning electron microscopy. Weddellite druses were made up of tetragonal crystallites, whereas those from whewellite were most often recognized by their acute points and general star-like shape. These studies clearly demonstrated that members from the main traditional subfamilies of the Cactaceae family could synthesize different chemical forms of calcium oxalate, suggesting a definite but different genetic control. The direct relationship established between a given Cactaceae species and a definite calcium oxalate biomineral seems to be a useful tool for plant identification and chemotaxonomy.     

The intravacuolar organic matrix associated with calcium oxalate crystals in leaves of Vitis

In Vitis (grape) calcium oxalate crystals form in a needle-like morphology unique to plants, presenting an intriguing system of biological control over mineral formation. Crystals develop within an organic matrix which appears to provide control over the sites and forms of crystal deposition; however, little is known about the chemical nature of the matrix. A procedure has been developed to isolate crystals along with their associated intravacuolar matrix from leaves of grape, and studies have been initiated into the chemical composition of the matrix by characterizing elemental content, carbohydrates, and protein. The isolated matrix consisted of two structural phases, membrane chambers enclosing developing crystals, and a water-soluble phase surrounding the crystal chambers. Elemental analysis detected substantial calcium and potassium, as well as some iron in the water-soluble phase. Analysis of the water-soluble matrix by GC-MS showed that it contained an unusual polymer with novel glucuronic acid linkages. In addition, linkage analysis indicated 5-linked arabinans, arabinogalactan, and various mannosyl units typical of complex carbohydrates of N-linked glycoproteins. SDS—PAGE analysis of the water-soluble matrix and crystal chambers showed that each had distinct banding profiles in silver-stained gels, with prominent 60 and 70 kDa polypeptides in crystal chamber extracts. Demineralization studies provided direct evidence that the isolated matrix promotes crystal nucleation. The findings about the organic matrix associated with calcium oxalate crystals in grape are discussed in relation to crystal nucleation and growth and features shared with animal and microbial biomineralization systems.     

Scanning electron microscopy of calcium oxalate on mantle hyphae of hybrid larch roots from a farm forestry experimental site

An examination of roots of hybrid larch from a farm forestry site by scanning electron microscopy has revealed crystalline deposits encrusting mantle hyphae of the associated ectomycorrhizal fungus. Electron probe micro-analysis identified calcium in the crystals and X-ray diffraction showed them to be whewellite, the monohydrate form of calcium oxalate. The significance of the finding is discussed.     

Characterization of histone (H1B) oxalate binding protein in experimental urolithiasis and bioinformatics approach to study its oxalate interaction

The rat kidney H1 oxalate binding protein was isolated and purified. Oxalate binds exclusively with H1B fraction of H1 histone. Oxalate binding activity is inhibited by lysine group modifiers such as 4',4'-diisothiostilbene-2,2-disulfonic acid (DIDS) and pyridoxal phosphate and reduced in presence of ATP and ADP. RNA has no effect on oxalate binding activity of H1B whereas DNA inhibits oxalate binding activity. Equilibrium dialysis method showed that H1B oxalate binding protein has two binding sites for oxalate, one with high affinity, other with low affinity. Histone H1B was modeled in silico using Modeller8v1 software tool since experimental structure is not available. In silico interaction studies predict that histone H1B-oxalate interaction take place through lysine121, lysine139, and leucine68. H1B oxalate binding protein is found to be a promoter of calcium oxalate crystal (CaOx) growth. A 10% increase in the promoting activity is observed in hyperoxaluric rat kidney H1B. Interaction of H1B oxalate binding protein with CaOx crystals favors the formation of intertwined calcium oxalate dehydrate (COD) crystals as studied by light microscopy. Intertwined COD crystals and aggregates of COD crystals were more pronounced in the presence of hyperoxalauric H1B.     

Production of Calcium Oxalate Crystals by the Basidiomycete Moniliophthora perniciosa, the Causal Agent of Witches’ Broom Disease of Cacao

Oxalic acid has been shown as a virulence factor for some phytopathogenic fungi, removing calcium from pectin and favoring plant cell wall degradation. Recently, it was published that calcium oxalate accumulates in infected cacao tissues during the progression of Witches’ Broom disease (WBD). In the present work we report that the hemibiotrophic basidiomycete Moniliophthora perniciosa, the causal agent of WBD, produces calcium oxalate crystals. These crystals were initially observed by polarized light microscopy of hyphae growing on a glass slide, apparently being secreted from the cells. The analysis was refined by Scanning electron microscopy and the compositon of the crystals was confirmed by energy-dispersive x-ray spectrometry. The production of oxalate by M. perniciosa was reinforced by the identification of a putative gene coding for oxaloacetate acetylhydrolase, which catalyzes the hydrolysis of oxaloacetate to oxalate and acetate. This gene was shown to be expressed in the biotrophic-like mycelia, which in planta occupy the intercellular middle-lamella space, a region filled with pectin. Taken together, our results suggest that oxalate production by M. perniciosa may play a role in the WBD pathogenesis mechanism.     

Oxalic Acid Has an Additional,Detoxifying Function in Sclerotinia sclerotiorum Pathogenesis

The mechanism of the diseases caused by the necrotroph plant pathogen Sclerotinia sclerotiorum is not well understood. To investigate the role of oxalic acid during infection high resolution, light-, scanning-, transmission electron microscopy and various histochemical staining methods were used. Our inoculation method allowed us to follow degradation of host plant tissue around single hyphae and to observe the reaction of host cells in direct contact with single invading hyphae. After penetration the outer epidermal cell wall matrix appeared degraded around subcuticular hyphae (12-24 hpi). Calcium oxalate crystals were detected in advanced (36-48 hpi) and late (72 hpi) infection stages, but not in early stages. In early infection stages, surprisingly, no toxic effect of oxalic acid eventually secreted by S. sclerotiorum was observed. As oxalic acid is a common metabolite in plants, we propose that attacked host cells are able to metabolize oxalic acid in the early infection stage and translocate it to their vacuoles where it is stored as calcium oxalate. The effects, observed on healthy tissue upon external application of oxalic acid to non-infected, living tissue and cell wall degradation of dead host cells starting at the inner side of the walls support this idea. The results indicate that oxalic acid concentrations in the early stage of infection stay below the toxic level. In plant and fungi oxalic acid/calcium oxalate plays an important role in calcium regulation. Oxalic acid likely could quench calcium ions released during cell wall breakdown to protect growing hyphae from toxic calcium concentrations in the infection area. As calcium antimonate-precipitates were found in vesicles of young hyphae, we propose that calcium is translocated to the older parts of hyphae and detoxified by building non-toxic, stable oxalate crystals. We propose an infection model where oxalic acid plays a detoxifying role in late infection stages.     

Calcium Oxalate Crystals in Developing Seeds of Soybean

Young developing soybean seeds contain relatively large amountsof calcium oxalate (CaOx) monohydrate crystals. A test for Caand CaOx indicated that Ca deposits and crystals initially occurredin the funiculus, where a single vascular bundle enters theseed. Crystals formed in the integuments until the embryo enlargedenough to crush the inner portion of the inner integument. Crystalsthen appeared in the developing cotyledon tissues and embryoaxis. All crystals formed in cell vacuoles. Dense bodies andmembrane complexes were evident in the funiculus. In the innerintegument, cell vacuoles assumed the shape of the future crystals.This presumed predetermined crystal mould is reported here forthe first time for soybean seeds. As crystals in each tissuenear maturity, a wall forms around each crystal. This intracellularcrystal wall becomes contiguous with the cell wall. Integumentcrystals remain visible until the enlarging embryo crushes theinteguments; the crystals then disappear. A related study revealedthat the highest percent of oxalate by dry mass was reachedin the developing +16 d (post-fertilization) seeds, and thendecreased during late seed maturation. At +60 d, CaOx formationand disappearance are an integral part of developing soybeanseeds. Our results suggest that Ca deposits and crystals functionallyserve as Ca storage for the rapidly enlarging embryos. The oxalate,derived from one or more possible metabolic pathways, couldbe involved in seed storage protein synthesis. Copyright 2001Annals of Botany Company Calcium, crystals, development, Glycine max, ovule, oxalate, seed, soybean     

Elucidation of multiple forms of nephrocalcin by P-NMR spectrometer

Four forms of nephrocalcin have been routinely isolated from mammalian kidney tissues and urine using DEAE-cellulose column chromatography with a linear NaCl gradient. We have demonstrated that these four forms of nephrocalcin, isolated from bovine kidneys, contain different amounts of phosphate residues, and that alkaline phosphatase digestion converts these to only one form of nephrocalcin. The changes in the nephrocalcin before and after removal of phosphate residues were measured by ³¹P-NMR spectrometer. Loss of phosphate residues decreased the dissociation constant of nephrocalcin 10-fold toward calcium oxalate monohydrate crystals, suggesting the phosphate residues appear to be important in the inhibitory effects of calcium oxalate monohydrate crystal growth.     

Uptake of calcium by the endoplasmic reticulum of the frog photoreceptor

We studied retinal photoreceptors of Rana pipiens by using techniques designed to investigate calcium localization. Particularly useful were methods in which intracellular sites of calcium uptake were detected by incubation of saponin-treated isolated retinas in calcium-containing media, with oxalate present as a trapping agent. With these procedures, cell compartments accumulate deposits, which can be shown to contain calcium by x-ray microanalysis. Calcium accumulation was prominent in the rough endoplasmic reticulum in the myoid region. In addition, deposits were observed in agranular reticulum and in certain Golgi- associated compartments of the myoid region, in mitochondria, in axonal reticulum, and in agranular reticulum of presynaptic terminals. Calcium was also detected in the endoplasmic reticulum of retinas fixed directly upon isolation, by a freeze-substitution method. The factors influencing accumulation of calcium in the endoplasmic reticulum were evaluated by a semiquantitative approach based on determining the relative frequency of calcium oxalate crystals under varying conditions. Calcium accumulation was markedly enhanced by ATP. Studies with a nonhydrolyzable ATP analogue (adenylyl- imidodiphosphate ) and with inhibitors of the sarcoplasmic reticulum Ca2+-Mg2+ ATPase (mersalyl and tetracaine) indicated that this ATP-dependent calcium uptake reflects an energy-dependent process roughly comparable to that in the sarcoplasmic reticulum.