Calcium channels are involved in calcium oxalate crystal formation in specialized cells of Pistia stratiotes L

BACKGROUND AND AIMS: Pistia stratiotes produces large amounts of calcium (Ca) oxalate crystals in specialized cells called crystal idioblasts. The potential involvement of Ca(2+) channels in Ca oxalate crystal formation by crystal idioblasts was investigated. METHODS: Anatomical, ultrastructural and physiological analyses were used on plants, fresh or fixed tissues, or protoplasts. Ca(2+) uptake by protoplasts was measured with (45)Ca(2+), and the effect of Ca(2+) channel blockers studied in intact plants. Labelled Ca(2+) channel blockers and a channel protein antibody were used to determine if Ca(2+) channels were associated with crystal idioblasts. KEY RESULTS: (45)Ca(2+) uptake was more than two orders of magnitude greater for crystal idioblast protoplasts than mesophyll protoplasts, and idioblast number increased when medium Ca was increased. Plants grown on media containing 1-50 microM of the Ca(2+) channel blockers, isradipine, nifedipine or fluspirilene, showed almost complete inhibition of crystal formation. When fresh tissue sections were treated with the fluorescent dihydropyridine-type Ca(2+) channel blocker, DM-Bodipy-DHP, crystal idioblasts were intensely labelled compared with surrounding mesophyll, and the label appeared to be associated with the plasma membrane and the endoplasmic reticulum, which is shown to be abundant in idioblasts. An antibody to a mammalian Ca(2+) channel alpha1 subunit recognized a single band in a microsomal protein fraction but not soluble protein fraction on western blots, and it selectively and heavily labelled developing crystal idioblasts in tissue sections. CONCLUSIONS: The results demonstrate that Ca oxalate crystal idioblasts are enriched, relative to mesophyll cells, in dihydropyridine-type Ca(2+) channels and that the activity of these channels is important to transport and accumulation of Ca(2+) required for crystal formation.     

Calcium oxalate formation is a rapid and reversible process inLemna minor L.

Summary Lemna minor root tips form raphide Ca oxalate crystals in both the root cap and root proper. An in vivo system was developed to examine raphide crystal bundle formation in the root of intact plants. By increasing the exogenous Ca concentration, crystal bundle formation could be induced. Entire new crystal bundles could be formed within 30 minutes of an inductive stimulus. The process was reversible with recently formed crystal bundles being dissolved over a period of about 3 hours. Older, previously existing bundles were more resistant to dissolution. The calmodulin antagonists, chlorpromazine and trifluoperazine (300 M), prevented crystal formation and caused dissolution of some crystal bundles, even in the presence of exogenous Ca. When the antagonists were flushed out and replaced with fresh medium, crystals were formed in cells where dissolution had occurred under the influence of the antagonists. The Ca ionophore A 23187 (20 M) caused slow dissolution of crystal bundles, even in the presence of exogenous Ca. A model describing the control of and physiological significance of Ca oxalate formation in plants is presented and discussed with respect to the results obtained in this study.     

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.     

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.     

Cell and calcium oxalate crystal growth is coordinated to achieve high-capacity calcium regulation in plants

Summary Crystal idioblasts are cells which are specialized for accumulation of Ca²⁺ as a physiologically inactive, crystalline salt of oxalic acid. Using microautoradiographic, immunological, and ultrastructural techniques, the process of raphide crystal growth, and how crystal growth is coordinated with cell growth, was studied in idioblasts ofPistia stratiotes. Incorporation of⁴⁵Ca²⁺ directly demonstrated that, relative to surrounding mesophyll cells, crystal idioblasts act as high-capacity Ca²⁺ sinks, accumulating large amounts of Ca²⁺ within the vacuole as crystals. The pattern of addition of Ca²⁺ during crystal growth indicates a highly regulated process with bidirectional crystal growth. In very young idioblasts,⁴⁵Ca²⁺ is incorporated along the entire length of the needle-shaped raphide crystals, but as they mature incorporation only occurs at crystal tips in a bidirectional mode. At full maturity, the idioblast stops Ca²⁺ uptake, although the cells are still alive, demonstrating an ability to strictly regulate Ca transport processes at the plasma membrane. In situ hybridization for ribosomal RNA shows young idioblasts are extremely active cells, are more active than older idioblasts, and have higher general activity than surrounding mesophyll cells. Polarizing and scanning electron microscopy demonstrate that the crystal morphology changes as crystals develop and includes morphological polarity and an apparent nucleation point from which crystals grow bidirectionally. These results indicate a carefully regulated process of biomineralization in the vacuole. Finally, we show that the cytoskeleton is important in controlling the idioblast cell shape, but the regulation of crystal growth and morphology is under a different control mechanism.Abbreviation SEM scanning electron microscopy     

Incorporation of strontium into plant calcium oxalate crystals

Summary Lemna minor, which produces many calcium oxalate raphide crystals, was grown on media containing in addition to Ca, 200 M of one of the following divalent cations: Ba, Cd, Co, Mn or Sr. Energy dispersive X-ray analysis showed that only Sr was incorporated into the raphides at levels detectable by the analysis technique. Incorporation of Sr into other insoluble compounds, such as cell wall material, could not be detected. Plant species which form different crystal types in their leaves (Beta vulgaris, crystal sand;Arthrostema ciliatum, druse;Glycine canescens, prismatic) also incorporated Sr into their crystals when grown hydroponically on nutrient medium containing 200 M Sr.Axenic cultures ofL. minor were used to examine further the process of Sr incorporation into plant crystals. When grown on nutrient solution with 5 M Ca, increasing the Sr concentration resulted in increases of the amount of Sr incorporated into the raphide crystals. The ratio of Sr to Ca became greater as the Sr concentration was increased. This ratio change was due to both an increase in the amount of Sr incorporated and a decrease in the Ca incorporated. Analysis of the number of crystal idioblasts formed as a function of Sr concentration shows fewer idioblasts are produced as Sr became high. Competition with Ca and interference of Ca utilization by Sr is indicated.     

DISTRIBUTION OF CALCIUM OXALATE CRYSTAL IDIOBLASTS IN LEAVES OF TARO (COLOCASIA ESCULENTA)

Cleared leaves of taro (Colocasia esculenta) were examined microscopically to determine changes in the distribution of both druse and raphide idioblasts during a late developmental process—leaf unfurling and expansion. Druse crystal idioblasts are small spherical cells found throughout the lamina, mostly in subepidermal areas. Two types of raphide idioblasts were observed in taro leaves: the nondefensive raphide idioblasts, which are elongated cells usually found embedded in tissues of the leaf margins; and the defensive raphide idioblasts, also elongated cells, but usually found suspended between mesophyll cells in leaf airspaces. The densities of both druse and raphide cells were highest at the fully furled stage and least in the mature, unfurled stage, after substantial leaf expansion. During leaf unfurling, the raphide cells showed a bilaterally symmetrical distribution during all stages from fully furled to mature, unfurled leaves. The distribution of druse cells was bilaterally symmetrical during the fully furled and unfurled stages, but, during unfurling, when one half of the lamina is unfurled and the other half is still tightly furled, up to 80% of the druse cells were found on the unfurled half of the lamina.     

Cellular ultrastructure and crystal development in Amorphophallus (Araceae)

Background and Aims: Species of Araceae accumulate calcium oxalate in the form ofcharacteristically grooved needle-shaped raphide crystals andmulti-crystal druses. This study focuses on the distributionand development of raphides and druses during leaf growth inten species of Amorphophallus (Araceae) in order to determinethe crystal macropatterns and the underlying ultrastructuralfeatures associated with formation of the unusual raphide groove. Methods: Transmission electron microscopy (TEM), scanning electron microscopy(SEM) and both bright-field and polarized-light microscopy wereused to study a range of developmental stages. Key Results: Raphide crystals are initiated very early in plant development.They are consistently present in most species and have a fairlyuniform distribution within mature tissues. Individual raphidesmay be formed by calcium oxalate deposition within individualcrystal chambers in the vacuole of an idioblast. Druse crystalsform later in the true leaves, and are absent from some species.Distribution of druses within leaves is more variable. Drusesinitially develop at leaf tips and then increase basipetallyas the leaf ages. Druse development may also be initiated incrystal chambers. Conclusions: The unusual grooved raphides in Amorphophallus species probablyresult from an unusual crystal chamber morphology. There aremultiple systems of transport and biomineralization of calciuminto the vacuole of the idioblast. Differences between raphideand druse idioblasts indicate different levels of cellular regulation.The relatively early development of raphides provides a defensivefunction in soft, growing tissues, and restricts build-up ofdangerously high levels of calcium in tissues that lack theability to adequately regulate calcium. The later developmentof druses could be primarily for calcium sequestration.     

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.     

Calcium Deposition in Idioblasts of Mulberry Leaves

Large, rounded idioblasts were observed in adaxial leaves ofmulberry plants; they were clearly distinguishable from epidermal,trichome and parenchyma cells. The size and density of idioblastsvaried according to leaf age. Cytological features of idioblastswere as follows: the outermost region (‘cap’) ofidioblasts was situated on the adaxial surface as a dome-likeprotrusion; a cylindrical protuberance extended from the capregion to the inner part of the idioblast; in idioblasts frommature leaves a crystal mass was suspended from the lower tipof the cylindrical protuberance. Elemental analysis of idioblastsdemonstrated that silicon (Si) was localized in both the capregion and the cylindrical protuberance but calcium (Ca) waspresent in the large crystal, indicating site-specific cellularlocalization of Ca and Si within an idioblast. Histochemicalassays showed that a distinct Ca crystal filled the vacuolesof idioblasts in mature leaves, while immature leaves had manyidioblasts without Ca deposition. The increase in the Ca contentof leaves was directly proportional to the increase in leafage and appeared to be closely related to the Ca sink capacityof the developing idioblast vacuoles. The maximum sink capacitywas quantified to be approximately 40 ng per idioblast whenmulberry plants were grown hydroponically with excess Ca.Copyright1999 Annals of Botany Company Morus alba, idioblast, Ca deposition, Ca sink capacity, silicon, X-ray microanalysis, histochemistry, scanning electron microscopy.     

Biosynthesis of L-ascorbic acid and conversion of carbons 1 and 2 of L-ascorbic acid to oxalic acid occurs within individual calcium oxalate crystal idioblasts

L-Ascorbic acid (AsA) and its metabolic precursors give rise to oxalic acid (OxA) found in calcium oxalate crystals in specialized crystal idioblast cells in plants; however, it is not known if AsA and OxA are synthesized within the crystal idioblast cell or transported in from surrounding mesophyll cells. Isolated developing crystal idioblasts from Pistia stratiotes were used to study the pathway of OxA biosynthesis and to determine if idioblasts contain the entire path and are essentially independent in OxA synthesis. Idioblasts were supplied with various (14)C-labeled compounds and examined by micro-autoradiography for incorporation of (14)C into calcium oxalate crystals. [(14)C]OxA gave heavy labeling of crystals, indicating the isolated idioblasts are functional in crystal formation. Incubation with [1-(14)C]AsA also gave heavy labeling of crystals, whereas [6-(14)C]AsA gave no labeling. Labeled precursors of AsA (L-[1-(14)C]galactose; D-[1-(14)C]mannose) also resulted in crystal labeling, as did the ascorbic acid analog, D-[1-(14)C]erythorbic acid. Intensity of labeling of isolated idioblasts followed the pattern OxA > AsA (erythorbic acid) > L-galactose > D-mannose. Our results demonstrate that P. stratiotes crystal idioblasts synthesize the OxA used for crystal formation, the OxA is derived from the number 1 and 2 carbons of AsA, and the proposed pathway of ascorbic acid synthesis via D-mannose and L-galactose is operational in individual P. stratiotes crystal idioblasts. These results are discussed with respect to fine control of calcium oxalate precipitation and the concept of crystal idioblasts as independent physiological compartments.     

Calcium and silica biomineralizations in leaves of eleven aquatic species of the Pampean Plain, Argentina

Calcium oxalate crystal and silicophytolith assemblages were investigated in leaves of eleven aquatic species from habitats of the Argentinean Pampean Plain. Calcium oxalate druses and raphide bundles mainly occurred in leaf parenchyma and aerenchyma. Druses were observed in Alternanthera philoxeroides, Ludwigia peploides, Polygonum hydropiperoides and Rumex crispus; and raphide bundles in L. peploides and Typha latifolia. No calcium oxalate crystals were observed in Bidens laevis, Mikania parodii, Solanum glaucophyllum, Ranunculus apiifolius or the Juncaceae family. Druse density ranged between 14.62 ± 3.09 (R. crispus) to 39.6 ± 7.2 crystals mm⁻² (P. hydropiperoides), and raphide bundle density ranged between 7.55 ± 1.36 (L. peploides) to 20.55 ± 5.19 crystals mm⁻² (T. latifolia). Silicification mainly occurred in epidermal cells and xylem. Juncus spp., S. glaucophyllum, B. laevis, M. parodii and R. apiifolius produced abundant and diverse phytolith morphotypes. The rest of the species produced very few (L. peploides, P. hydropiperoides, A. philoxeroides) or no identifiable phytoliths (T. latifolia, R. crispus). Silica content ranged from 0.07 (T. latifolia) to 4.7% dry weight (S. glaucophyllum).     

Calcium Oxalate Deposits in Leaves of Corchorus olitorius as Related to Accumulation of Toxic Metals1

This study was to report and describe the formation of Ca oxalate crystals and to explore whether there is any correlation between their abundant formation and the ability of plant to uptake and accumulate high levels of toxic metals. Soil-grown Corchorus olitorius L. (Tiliaceae) seedlings were further grown in water culture in the presence of Cd, Pb, Cu, or Al (0–10 g/ml) for 20 days. Light and electron microscopic examinations revealed a large number of intracellular prismatic-shaped Ca oxalate crystals in both leaf and callus cells. Crystals were formed in the vacuole, a single large crystal being formed per cell. The crystal-containing cells differed in size and shape from crystal-free cells, they were rich in organelles, membranes, and vesicles and have dense cytoplasm, enlarged nucleus and modified starch-lacking plastids with few grana. These cells look highly active. Corchorus plants treated with Cd, Pb, Cu, and Al accumulated these metals to the levels several times higher than untreated plants. The contents of Pb, Cd, Al, and Cu in leaf tissues of plants grown in the presence of 5 g/ml of these metals were 10, 20, 25, and 40 times higher, respectively, than those in plants grown on media devoid of them. X-ray microanalysis of Ca oxalate crystals in leaves from plants exposed to 5 g/ml Cd, Pb, Al, or Cu indicated the incorporation only of Al into these crystals. Results of this paper suggest a possible contribution for Ca oxalate-crystal formation in sequestering and tolerance of at least some toxic metals.     

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     

Absence of CeCl3-detectable peroxisomal glycolate-oxidase activity in developing raphide crystal idioblasts in leaves of Psychotria punctata Vatke and roots of Yucca torreyi L.

Three peroxisomal enzymes, glycolate oxidase, urate oxidase and catalase were localized cytochemically in Psychotria punctata (Rubiaceae) leaves and Yucca torreyi (Agavaceae) seedling root tips, both of which contain developing and mature calcium-oxalate raphide crystal idioblasts. Glycolate-oxidase (EC 1.1.3.1) and catalase (EC 1.11.1.6) activities were present within leaftype peroxisomes in nonidioblastic mesophyll cells in Psychotria leaves, while urate-oxidase (EC 1.7.3.3) activity could not be conclusively demonstrated in these organelles. Unspecialized peroxisomes in cortical parenchyma of Yucca roots exhibited activities of all three enzymes. Reactionproduct deposits attributable to glycolate-oxidase activity were never observed in peroxisomes of any developing or mature crystal idioblasts of Psychotria or Yucca. Catalase localization indicates that idioblast microbodies are functional peroxisomes. The apparent absence of glycolate oxidase in crystal idioblasts of Psychotria and Yucca casts serious doubt that pathways involving this enzyme are operational in the synthesis of the oxalic acid precipitated as calcium-oxalate crystals in these cells.Abbreviations AMPD 2-amino-2-methyl-1,3-propandiol - CTEM conventional transmission electron microscopy - DAB 3,3-diaminobenzidine tetrahydrochloride - HVEM high-voltage electron microscopy     

The Occurrence, Type and Location of Calcium Oxalate Crystals in the Leaves of Fourteen Species of Araceae

The occurrence, type and location of calcium oxalate crystalsin the leaves of 14 species belonging to the family Araceaewere studied by light microscopy. The Pizzolato test and theRubeanic acid-silver nitrate test, used to chemically identifyand locate the crystals in cross sections of laminae, showedthe presence of four types of crystals: druses, raphides, prismaticsand crystal sand. Styloids were not observed in any of the species.Crystals identified as calcium oxalate were observed in eachtissue layer of the leaf blade, druses occurring more frequentlyin the palisade mesophyll layers, raphides more often in thespongy mesophyll. Prismatics were sparse, occurring in the mesophyllof only two species. Specialized spindle-shaped crystal idioblasts,located in the spongy mesophyll in all cases, were observedin seven of the 14 aroids. Crystal sand and variations in crystalforms were most frequently observed to be calcium compoundsother than calcium oxalate. Crystals, calcium oxalate, idioblasts, Araceae     

天津盐渍化生境54种植物钙晶体与钙组分特征

植物钙包括游离态的Ca²⁺和结合态易溶、微溶和难溶于水的钙盐,而难溶于水的钙盐常会形成钙晶体.为了解盐渍化生境中不同生长型植物体内的钙状况,本文对天津市54种植物进行了钙晶体的镜检和钙组分的测定.结果表明： 在盐渍化生境中的54种植物体内,有38种植物体内镜检到较多的钙晶体,其中37种植物体内为以簇晶和方晶为主的草酸钙晶体,只在桑科的无花果叶片中观察到内含碳酸钙晶体的钟乳体.按生长型统计,落叶乔、灌木体内的草酸钙晶体较多,藤本植物体内的草酸钙晶体较少,而草本植物和常绿乔木体内未镜检到草酸钙晶体.同时,从乔木、灌木、藤本到草本,植物体内盐酸溶性钙含量逐渐减少而水溶性钙含量逐渐增多,且草本植物体内的水溶性钙含量显著高于乔木和灌木.在盐渍化生境中,植物体内的钙晶体和钙组分因生长型不同而有所差异,草酸钙在落叶乔、灌木抵御盐分胁迫中发挥着重要作用.     

Oxalic acid metabolism and calcium oxalate formation in Lemna minor L.

Abstract Axenic Lemna minor plants, which form numerous calcium oxalate crystals, were exposed to [¹⁴C]-glycolic acid, -glyoxylic acid, -oxalic acid and -ascorbic acid and prepared for microautoradiography by a technique that preserves only insoluble label to determine specifically the pathway leading to oxalic acid used for crystal formation. Label from glycolic, glyoxylic, and oxalic acids was incorporated into crystals. Label from oxalic acid was also found in starch when exposure to label was done in the light but not dark, while plastids specialized for lipid storage were heavily labelled under both conditions. Incorporation of label from glycolic and glyoxylic acids, but not oxalic acid, was inhibited in the presence of the glycolate oxidase inhibitors, αHPMS (2-pyridylhydroxy methanesulphonic acid) and mHBA (methyl 2-hydroxy-3-butynoic acid), and inhibition of labelling was not due to an effect on uptake. These studies show that the glycolate oxidase pathway to oxalic acid is operational in L. minor and that the product is available for crystal formation. Dark-grown plants form almost four times as many crystal cells (idioblasts) as do light-grown plants, indicating crystal formation is not in response to photorespiratory glycolate production. Label from [1-¹⁴C]ascorbic acid was also incorporated into crystals and labelling was inhibited by mHBA, indicating glycolic acid and/or glyoxylic acid are possible intermediates of ascorbic acid catabolism. The effect of nitrogen source on crystal formation was also investigated. Significantly more crystal idioblasts were formed, on a surface area basis, by plants grown on ammonium than by plants grown on nitrate nitrogen. When grown with mixed ammonium and nitrate, an intermediate number of crystal idioblasts were formed.     

Calcium,Calcium Oxalate Crystals,and Leaf Differentiation in the Common Bean (Phaseolus vulgaris L.)*

Young plants of Phaseolus vulgaris were grown in nutrient solutions at different levels of calcium concentration. When the calcium concentration was low more palisade parenchyma and less extended bundle sheath was formed at the adaxial side of minor veins of the leaves as compared to leaves of plants grown with higher calcium supply. The number of calcium oxalate crystals in the bundle sheath extensions was positively correlated to the amount of calcium fed to the plants. The ion induces additional cell divisions in the bundle sheath extensions. A high supply of calcium leads to the formation of a second type of crystal in the bundle sheath.     

Cell wall sheath surrounding calcium oxalate crystals in mulberry idioblasts

Summary. The distribution and ultrastructural features of idioblasts containing calcium oxalate crystals were studied in leaf tissues of mulberry, Morus alba L. In addition to the calcium carbonate crystals formed in epidermal idioblasts, large calcium oxalate crystals were deposited in cells adjacent to the veins and surrounded by a cell wall sheath which had immunoreactivity with an antibody recognizing a xyloglucan epitope. The wall sheath formation indicates exclusion of the mature crystal from the protoplast. Correspondence: Y. Sugimura, Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido, Matsugasaki, Sakyo, Kyoto 606-8585, Japan.