Isolation of Medicago truncatula mutants defective in calcium oxalate crystal formation

Plants accumulate crystals of calcium oxalate in a variety of shapes, sizes, amounts, and spatial locations. How and why many plants form crystals of calcium oxalate remain largely unknown. To gain insight into the regulatory mechanisms of crystal formation and function, we have initiated a mutant screen to identify the genetic determinants. Leaves from a chemically mutagenized Medicago truncatula population were visually screened for alterations in calcium oxalate crystal formation. Seven different classes of calcium oxalate defective mutants were identified that exhibited alterations in crystal nucleation, morphology, distribution and/or amount. Genetic analysis suggested that crystal formation is a complex process involving more than seven loci. Phenotypic analysis of a mutant that lacks crystals, cod 5, did not reveal any difference in plant growth and development compared with controls. This finding brings into question the hypothesized roles of calcium oxalate formation in supporting tissue structure and in regulating excess tissue calcium.     

Engineering calcium oxalate crystal formation in Arabidopsis

Many plants accumulate crystals of calcium oxalate. Just how these crystals form remains unknown. To gain insight into the mechanisms regulating calcium oxalate crystal formation, a crystal engineering approach was initiated utilizing the non-crystal-accumulating plant, Arabidopsis. The success of this approach hinged on the ability to transform Arabidopsis genetically into a calcium oxalate crystal-accumulating plant. To accomplish this transformation, two oxalic acid biosynthetic genes, obcA and obcB, from the oxalate-secreting phytopathogen, Burkholderia glumae were inserted into the Arabidopsis genome. The co-expression of these two bacterial genes in Arabidopsis conferred the ability not only to produce a measurable amount of oxalate but also to form crystals of calcium oxalate. Biochemical and cellular studies of crystal accumulation in Arabidopsis revealed features that are similar to those observed in the cells of crystal-forming plants. Thus, it appears that at least some of the basic components that comprise the calcium oxalate crystal formation machinery are conserved even in non-crystal-accumulating plants.     

Oxalobacter formigenes and its role in oxalate metabolism in the human gut

Oxalate is ingested in a wide range of animal feeds and human foods and beverages and is formed endogenously as a waste product of metabolism. Bacterial, rather than host, enzymes are required for the intestinal degradation of oxalate in man and mammals. The bacterium primarily responsible is the strict anaerobe Oxalobacter formigenes. In humans, this organism is found in the colon. O. formigenes has an obligate requirement for oxalate as a source of energy and cell carbon. In O. formigenes, the proton motive force for energy conservation is generated by the electrogenic antiport of oxalate(2-) and formate(1-) by the oxalate-formate exchanger, OxlT. The coupling of oxalate-formate exchange to the reductive decarboxylation of oxalyl CoA forms an 'indirect' proton pump. Oxalate is voided in the urine and the loss of O. formigenes may be accompanied by elevated concentrations of urinary oxalate, increasing the risk of recurrent calcium oxalate kidney stone formation. Links between the occurrence of nephrolithiasis and the presence of Oxalobacter have led to the suggestion that antibiotic therapy may contribute to the loss of this organism from the colonic microbiota. Studies in animals and human volunteers have indicated that, when administered therapeutically, O. formigenes can establish in the gut and reduce the urinary oxalate concentration following an oxalate load, hence reducing the likely incidence of calcium oxalate kidney stone formation. The findings to date suggest that anaerobic, colonic bacteria such as O. formigenes, that are able to degrade toxic compounds in the gut, may, in future, find application for therapeutic use, with substantial benefit for human health and well-being.     

An Assessment of Engineered Calcium Oxalate Crystal Formation on Plant Growth and Development as a Step toward Evaluating Its Use to Enhance Plant Defense

The establishment of new approaches to control chewing insects has been sought not only for direct use in reducing crop loss but also in managing resistance to the pesticides already in use. Engineered formation of calcium oxalate crystals is a potential strategy that could be developed to fulfill both these needs. As a step toward this development, this study investigates the effects of transforming a non-calcium oxalate crystal accumulating plant, Arabidopsis thaliana, into a crystal accumulating plant. Calcium oxalate crystal accumulating A. thaliana lines were generated by ectopic expression of a single bacterial gene encoding an oxalic acid biosynthetic enzyme. Biochemical and cellular studies suggested that the engineered A. thaliana lines formed crystals of calcium oxalate in a manner similar to naturally occurring crystal accumulating plants. The amount of calcium oxalate accumulated in leaves also reached levels similar to those measured in the leaves of Medicago truncatula in which the crystals are known to play a defensive role. Visual inspection of the different engineered lines, however, suggested a phenotypic consequence on plant growth and development with higher calcium oxalate concentrations. The restoration of a near wild-type plant phenotype through an enzymatic reduction of tissue oxalate supported this observation. Overall, this study is a first to provide initial insight into the potential consequences of engineering calcium oxalate crystal formation in non-crystal accumulating plants.     

Nucleation of calcium oxalate crystals on an imprinted polymer surface from pure aqueous solution and urine

Calcium oxalate (CaOx) is the most common component of human kidney stones. Heterogeneous nucleation is regarded as the key mechanism in this process. In this study, we have used an imprinted 6-methacrylamidohexanoic acid/divinylbenzene co-polymer as a biomimetic surface to nucleate CaOx crystal formation. The polymer was imprinted with either calcium oxalate monohydrate (COM) or dihydrate (COD) template crystals. These were washed out of the polymer, which was then immersed in various test solutions. The test solutions were an aqueous solution of calcium chloride and sodium oxalate, artificial urine and a sample of real urine. Crystals that formed on the polymer surface were characterized by X-ray powder diffraction, Fourier transform infrared spectroscopy, atomic absorption spectroscopy and scanning electron microscopy. Results showed that in the aqueous solution the COM-imprinted polymer induced the nucleation of COM. The COD-imprinted polymer induced only trace amounts of COD crystallization, together with larger quantities of COM. In artificial and real urines, COM also specifically precipitated on the COM-imprinted surface. The results show that, at least to some extent, the imprinted polymers direct formation of their morphologically matched crystals. In the case of COD, however, it appears that either rapid hydrate transformation of COD to COM occurs, or the more stable COM polymorph is directly co-precipitated by the polymer. Our results support the hypothesis that heterogeneous nucleation plays a key role in CaOx stone formation and that the imprinted polymer model could provide an additional and superior diagnostic tool for stone researchers to assess stone-risk in urine.Abbreviations COD calcium oxalate dihydrate - COM calcium oxalate monohydrate - COT calcium oxalate trihydrate - dvb divinylbenzene - 6-maaha 6-methylacrylamidohexanoic acid     

Medicago truncatula mutants demonstrate the role of plant calcium oxalate crystals as an effective defense against chewing insects

Calcium oxalate is the most abundant insoluble mineral found in plants and its crystals have been reported in more than 200 plant families. In the barrel medic Medicago truncatula Gaertn., these crystals accumulate predominantly in a sheath surrounding secondary veins of leaves. Mutants of M. truncatula with decreased levels of calcium oxalate crystals were used to assess the defensive role of this mineral against insects. Caterpillar larvae of the beet armyworm Spodoptera exigua Hübner show a clear feeding preference for tissue from calcium oxalate-defective (cod) mutant lines cod5 and cod6 in choice test comparisons with wild-type M. truncatula. Compared to their performance on mutant lines, larvae feeding on wild-type plants with abundant calcium oxalate crystals suffer significantly reduced growth and increased mortality. Induction of wound-responsive genes appears to be normal in cod5 and cod6, indicating that these lines are not deficient in induced insect defenses. Electron micrographs of insect mouthparts indicate that the prismatic crystals in M. truncatula leaves act as physical abrasives during feeding. Food utilization measurements show that, after consumption, calcium oxalate also interferes with the conversion of plant material into insect biomass during digestion. In contrast to their detrimental effects on a chewing insect, calcium oxalate crystals do not negatively affect the performance of the pea aphid Acyrthosiphon pisum Harris, a sap-feeding insect with piercing-sucking mouthparts. The results confirm a long-held hypothesis for the defensive function of these crystals and point to the potential value of genes controlling crystal formation and localization in crop plants.     

Herbivory and Calcium Concentrations Affect Calcium Oxalate Crystal Formation in Leaves ofSida (Malvaceae)

Calcium oxalate crystals have potential roles in plants as partof a defence mechanism against herbivores and/or in accumulatingexcess calcium. To date, these potential roles have been studiedindependently. In this experimental study the effects of calciumlevels and herbivory on the production of calcium oxalate crystals(i.e. druse, spherical crystal aggregates) were examined inseedlings of Sida rhombifolia. Seedlings were subjected to threecalcium levels (low, normal or high) and an artificial herbivorytreatment. Calcium levels and herbivory both affected densityof crystals in leaves. Leaves from seedlings grown in low calciumhad a greater crystal density than those grown in high calcium.Leaves from seedlings subjected to herbivory had a greater crystaldensity than those from seedlings not subjected to herbivory.This study provides additional evidence that calcium oxalatecrystal production depends not only on calcium levels but canalso be influenced by external pressures such as herbivory.In addition to their physiological role in plants, these resultssuggest that calcium oxalate crystals can also act as a defencemechanism against herbivores. Copyright 2001 Annals of BotanyCompany Calcium concentrations, calcium oxalate crystals, herbivory, Malvaceae, Sida rhombifolia     

Sialylation of urinary prothrombin fragment 1 is implicated as a contributory factor in the risk of calcium oxalate kidney stone formation

Urinary glycoproteins are important inhibitors of calcium oxalate crystallization and adhesion of crystals to renal cells, both of which are key mechanisms in kidney stone formation. This has been attributed to glycosylation of the proteins. In South Africa, the black population rarely form stones (incidence < 1%) compared with the white population (incidence 12-15%). A previous study involving urinary prothrombin fragment 1 from both populations demonstrated superior inhibitory activity associated with the protein from the black group. In the present study, we compared N-linked and O-linked oligosaccharides released from urinary prothrombin fragment 1 isolated from the urine of healthy and stone-forming subjects in both populations to elucidate the relationship between glycosylation and calcium oxalate stone pathogenesis. The O-glycans of both control groups and the N-glycans of the black control samples were significantly more sialylated than those of the white stone-formers. This demonstrates a possible association between low-percentage sialylation and kidney stone disease and provides a potential diagnostic method for a predisposition to kidney stones that could lead to the implementation of a preventative regimen. These results indicate that sialylated glycoforms of urinary prothrombin fragment 1 afford protection against calcium oxalate stone formation, possibly by coating the surface of calcium oxalate crystals. This provides a rationale for the established roles of urinary prothrombin fragment 1, namely reducing the potential for crystal aggregation and inhibiting crystal-cell adhesion by masking the interaction of the calcium ions on the crystal surface with the renal cell surface along the nephron.     

Morphologies and elemental compositions of calcium crystals in phyllodes and branchlets of Acacia robeorum (Leguminosae: Mimosoideae)

Background and Aims

Formation of calcium oxalate crystals is common in the plant kingdom, but biogenic formation of calcium sulfate crystals in plants is rare. We investigated the morphologies and elemental compositions of crystals found in phyllodes and branchlets of Acacia robeorum, a desert shrub of north-western Australia.

Methods

Morphologies of crystals in phyllodes and branchlets of A. robeorum were studied using scanning electron microscopy (SEM), and elemental compositions of the crystals were identified by energy-dispersive X-ray spectroscopy. Distributional patterns of the crystals were studied using optical microscopy together with SEM.

Key Results

According to the elemental compositions, the crystals were classified into three groups: (1) calcium oxalate; (2) calcium sulfate, which is a possible mixture of calcium sulfate and calcium oxalate with calcium sulfate being the major component; and (3) calcium sulfate · magnesium oxalate, presumably mixtures of calcium sulfate, calcium oxalate, magnesium oxalate and silica. The crystals were of various morphologies, including prisms, raphides, styloids, druses, crystal sand, spheres and clusters. Both calcium oxalate and calcium sulfate crystals were observed in almost all tissues, including mesophyll, parenchyma, sclerenchyma (fibre cells), pith, pith ray and cortex; calcium sulfate · magnesium oxalate crystals were only found in mesophyll and parenchyma cells in phyllodes.

Conclusions

The formation of most crystals was biologically induced, as confirmed by studying the crystals formed in the phyllodes from seedlings grown in a glasshouse. The crystals may have functions in removing excess calcium, magnesium and sulfur, protecting the plants against herbivory, and detoxifying aluminium and heavy metals.     

植物晶体的形态结构、生物功能及形成机制研究进展

晶体是植物体内产生的一种具有特殊形态结构与生理功能的代谢物,其分布广泛,已在500多种植物中发现有晶体的存在。晶体形态多样,有针晶、柱晶、棱晶、砂晶、簇晶等;类型丰富,有草酸钙晶体、钟乳体、硅质体、硫酸钙晶体及其它类型的晶体;功能特殊,具有钙调节、植物保护和防御、重金属解毒、离子平衡、缓解逆境胁迫及其它多种生物功能。晶体的形成涉及钙离子的体外吸收和体内转运,草酸的生物合成,以及钙离子和草酸的耦合过程;晶体的生长发育涉及液泡、晶异细胞的调控及与其它细胞结构的相互协作。对晶体的研究进行综述,以期为晶体的进一步研究提供基础资料。     

Fungal biotransformation of zinc silicate and sulfide mineral ores

In this work, several fungi with geoactive properties, including Aspergillus niger, Beauveria caledonica and Serpula himantioides, were used to investigate their potential bioweathering effects on zinc silicate and zinc sulfide ores used in zinc extraction and smelting, to gain understanding of the roles that fungi may play in transformations of such minerals in the soil, and effects on metal mobility. Despite the recalcitrance of these minerals, new biominerals resulted from fungal interactions with both the silicate and the sulfide, largely resulting from organic acid excretion. Zinc oxalate dihydrate was formed through oxalate excretion by the test fungi and the mineral surfaces showed varying patterns of bioweathering and biomineral formation. In addition, calcium oxalate was formed from the calcium present in the mineral ore fractions, as well as calcite. Such metal immobilization may indicate that the significance of fungi in effecting metal mobilization from mineral ores such as zinc silicate and zinc sulfide is rather limited, especially if compared with bacterial sulfide leaching. Nevertheless, important bioweathering activities of fungi are confirmed which could be of local significance in soils polluted by such materials, as well as in the mycorrhizosphere.     

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.     

Right place,right time: Spatiotemporal light regulation of plant growth and development

Recent advances in our understanding of the roles of photoreceptors in light-dependent regulation of plant growth and development have been rapid and significant. Developments have been reported for numerous plant photoreceptor signaling pathways, yet researchers have made the most progress in increasing our comprehension of the roles of phytochrome family members, as well as the intracellular roles of phytochromes and phytochrome-interacting proteins in light-dependent signaling. An understudied, but vitally important, area of phytochrome biology centers on the roles phytochromes play in intercellular and interorgan signaling at the molecular level that results in the coordination of growth responses between distinct tissues and organs. This frontier of research into the spatiotemporal roles of phytochromes, and more generally plant photoreceptors, which is only beginning to be investigated and understood at the molecular genetic level, has a rich history of physiological data.Key words: cryptochrome, photomorphogenesis, photoreception, photoreceptor, phytochrome, spatiotemporal     

Oxalate, calcium uptake and ATPase activity of sarcoplasmic reticulum vesicles.

Ca++-uptake and Mg++-Ca++-dependent ATPase activity of skeletal muscle sarcoplasmic reticulum vesicles were reciprocally affected by increasing the oxalate concentration from 0 to 4 mM. At 0-0.1 mM oxalate approximately 17% of the calcium was removed by the vesicles from the medium while the ATPase activity was maximal (approximately 0.66 mumoles Pi mg-1 protein min-1). Between 0.1 to 0.2 mM oxalate the ATPase activity was reduced to one-fifth but the uptake rose sharply and 100% of the 45Ca++ was removed from the medium. The uptake was maintained at this level at oxalate concentrations greater than 0.4 mM but the ATPase activity remained inhibited. The kinetics of Ca++-uptake and ATPase activity were also differentially affected by oxalate. In the presence of oxalate, ruthenium red had only a very slight inhibitory effect on the calcium uptake. Addition of 0.1 mM EGTA removed 80% of the Ca++ from preloaded vesicles within 10 min. The formation of insoluble Ca-oxalate salt on the surface of the vesicle is suggested by these results. Calculations based on the Ksp of the calcium oxalate salt are presented to show its formation and the possible speciation of a Ca-oxalate complex which may affect the Ca++-uptake and ATPase activity.     

Matrix Proteins in the Outer Shells of Molluscs

The shells of molluscs are composed mainly of calcium carbonate crystals, with small amounts of matrix proteins. For more than 50 years, they have attracted attention for their unique mechanical and biological properties. Only recently, however, have researchers begun to realize that it is the matrix proteins that control the formation of calcium carbonate crystals and play key roles in their extraordinary properties, despite the fact that matrix proteins comprise less than 5% of the shell weight. This article reviews the matrix proteins identified to date from the shells of molluscs, their structural characteristics, and their roles in shell formation. Some suggestions are given for further investigation based on the summary and analysis.     

The association of different urinary proteins with calcium oxalate hydromorphs. Evidence for non-specific interactions

It has been proposed that various urinary proteins interact specifically with different calcium oxalate hydromorphs and these interactions have important implications regarding the understanding of the onset and progress of kidney stone disease. Calcium oxalate monohydrate and dihydrate crystals were grown and characterised thoroughly to establish sample purity. These crystals were then incubated in artificial urine samples containing isolated urinary macromolecules. Crystal growth was prevented by saturating the incubation mix with calcium oxalate, and this was confirmed through electron microscopy and calcium measurements of the incubation mix. The surface interactions between the different calcium oxalate hydrates and urinary proteins were investigated by the use of Western blots and immunoassays. The same proteins, notably albumin, Tamm-Horsfall protein, osteopontin and prothrombin fragment 1, associated with both hydrates. There was a trend for more protein to associate with calcium oxalate dihydrate, and greater quantities of different proteins associated with both hydrates when Tamm-Horsfall protein was removed from the incubation mix. There is no evidence from this study to indicate that particular proteins interact with specific calcium oxalate hydrates, which in turn suggests that these protein-mineral interactions are likely to be mediated through non-specific charge interactions.     

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.     

Isolation of plant nuclei suitable for flow cytometry from recalcitrant tissue by use of a filtration column

Flow cytometry is widely applied in the determination of nuclear DNA content and ploidy level in many organisms. However, a difficulty with flow cytometry is the method's intrinsic inability to tolerate large particles that associate with the isolated nuclei. A suspension of plant nuclei can often contain a high level of crystalline calcium oxalate, which blocks the fluidics system of the flow cytometer. We designed a cotton column and added polyvinylpyrrolidone-40 to the buffer to remove phenolic impurities and cytoplasmic compounds from plant nuclei, making the suspension suitable for flow cytometry. This simple and highly efficient protocol enables isolation of intact nuclei from plant tissues containing high levels of polysaccharides, calcium oxalate crystals and other metabolites. Our protocol resulted in the isolation of intact nuclei from mature orchid leaves. This method can be used on recalcitrant tissues and is particularly effective on plants containing calcium oxalate crystals.