Calcium oxalate crystals in the red algaAntithamnion kylinii (Ceramiales): cytoplasmic and limited to indeterminate axes

Summary The red alga,Antithamnion kylinii Gardner, was found to have needle-shaped inclusions about 10 m long and less than 0.4 m thick. They ranged in abundance from one or a few in young cells to hundreds in fully enlarged cells. Under polarized light, the inclusions were birefringent, indicating crystallinity. Solubility tests suggested that the inclusions were composed of calcium oxalate: they dissolved in 1 N hydrochloric acid and in a saturated solution of aqueous cupric acetate, but they were not soluble in 10 N acetic acid or 5.25% sodium hypochlorite. X-ray microanalysis confirmed the presence of calcium. Calcium oxalate crystals were present in cells of indeterminate axes, but cells of determinate lateral filaments lacked them. Light and electron microscopic study demonstrated that the crystals were associated with the parietal cytoplasm. Calcium oxalate crystals were also present inA. defectum Kylin, but they were not found in ten more distantly related taxa.     

FINE STRUCTURE OF CAPITULAR FILAMENTS IN THE COENOCYTIC GREEN ALGA PENICILLUS1,2,3

Capitular filaments of Penicillus capitatus contain a large central vacuole. The parietal cytoplasm is densely packed, devoid of chloroplasts in the growing tip, and becomes convoluted and sponge-like as extensions of the vacuole penetrate the cytoplasm in mature portions of the filament. Structure of organelles and their distribution in the filament are described. The vacuole contains a variety of inclusions, such as membranous configurations, spherical bodies, electron dense bodies, and calcium oxalate monohydrate crystals, each of the latter surrounded by a chamber associated with microtubules. Endophytic bacteria are present throughout the vacuole and occasionally in the tip cytoplasm. Some vacuolar components of P. pyriformis are described for comparison.     

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     

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.     

Cytochemische Lokalisation von Kalzium im Mantelepithel von Lymnaea stagnalis (Gastropoda)

Zusammenfassung Mit Oxalat wurde im äußeren Mantelepithel von Lymnaea stagnalis Kalzium gefällt und das Präzipitat im Elektronenmikroskop untersucht. Durch Kontroll- und Lösungsversuche wurde die Spezifität der Methode verdeutlicht.Im Drüsenpolster war äußerst wenig, im Manteldachepithel viel Niederschlag. Letzteres scheint demnach den Hauptanteil des Ca auszuscheiden.Kalziumoxalatkristalle fanden sich außerhalb der Zellen nur im extrapallealen Raum, nicht jedoch in den Interzellularen. Das Kalzium wird durch die Zellen transportiert; es tritt dort sowohl frei im Zellplasma, als auch in Vesikeln auf.

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Cytochemical localization of calcium in the mantle epithelium of Lymnaea stagnalis

Summary Calcium was precipitated by the oxalate method and crystals were localized by electron microscopy in the outer mantle epithelium of Lymnaea stagnalis. The specificy of the method was demonstrated by controls and solubility tests.The supramarginal ridge contained extremely little precipitate in contrast to the epithelium of the mantle roof. Therefore it seems that this represents the major site of calcium secretion.Outside the cells crystals of calcium oxalate were found only in the extrapalleal space, not in the intercellular gaps. Calcium is transported through the cells, where it appears free in the cytoplasm as well as enclosed in vesicles.

     

Calcium oxalate crystals in the mature seeds of Norway spruce, Picea abies (L.) Karst.

 The morphology and location of crystals encountered in the mature seeds of Norway spruce, Picea abies (L.) Karst., were examined using light and field emission scanning electron microscopy (FESEM). Crystals of various forms and sizes were discovered in different regions and tissues of seeds, particularly in the testa and the nucellus. Both solitary crystals and druses were occasionally enveloped by protrusions of the megaspore membranes or the cuticle of the megagametophyte. Histological studies and acid solubility tests coupled with analysis using energy dispersive X-ray microanalysis and X-ray diffraction evinced the crystals as calcium oxalate, but were unable to identify different hydration forms. Calcium oxalate crystals were most abundant in the damaged and infected tissues, and in the structures that desiccate during the development of the seed. Based on these observations we concluded that the accumulation of calcium oxalate is a regular process belonging to maturation and defense mechanism in spruce seeds. Received: 1 July 1998 / Accepted: 20 September 1998     

香樟叶肉含晶细胞季节变化研究

为探讨香樟(Cinnamomum camphora)叶肉含晶细胞超微结构的季节变化,阐明香樟叶肉中草酸钙晶体在春夏秋冬的变化规律。该研究以多年生香樟(C. camphora)叶片为材料,分别于春夏秋冬四个季节露地取样,制作超薄切片,用透射电子显微镜(TEM)观察叶肉含晶细胞超微结构的变化。结果表明:春季时香樟叶肉中只有少数细胞有草酸钙晶体,数量较少,晶体结构多为柱状晶、方晶; 夏季时香樟叶肉细胞中随机分布于液泡的草酸钙晶体明显比春季的数量多、体积大、形态丰富,晶体多为柱状晶、方晶、针晶、簇晶; 秋季时香樟叶肉细胞草酸钙晶体和夏季的类似,数量较多,形态多样,以方晶和柱状晶针晶为主,伴有晶簇; 冬季时香樟叶肉含晶细胞晶体形态为柱状晶、方晶、针晶,数量比夏季和秋季的数量略有减少。该研究结果表明在一年四季中香樟叶肉细胞液泡中均有草酸钙晶体结构存在。     

五种C4荒漠植物光合器官中含晶细胞的比较分析

 为了探讨荒漠植物适应干旱环境的机理, 选择光合器官发生很大变化的5种C₄荒漠植物进行了解剖结构的对比研究。结果表明, 这5种 植物中含晶细胞的数量、大小、形态和分布位置等存在差异。白梭梭(Haloxylon persicum)和梭梭(H. ammodendron)的同化枝普遍具有含晶细 胞; 沙拐枣(Calligonum mongolicum)的含晶细胞很少, 一般只分布在贮水组织或靠近栅栏组织处; 木本猪毛菜(Salsola arbuscula)的含晶细 胞也不多, 主要分布在栅栏组织和表皮细胞之间; 猪毛菜(S. collina)的含晶细胞更少, 仅在贮水组织中偶尔可见晶簇。比较梭梭、白梭梭和 沙拐枣同化枝不同部位的解剖结构发现, 梭梭同化枝基部含晶细胞最多, 中部次之, 顶部最少; 白梭梭同化枝顶部的含晶细胞数量较多, 中部 及基部较少; 沙拐枣同化枝顶部与基部的粘液细胞较多, 中部较少, 基部几乎没有栅栏组织, 而其维管组织较为发达。综合晶体的酸碱溶解性 及硝酸银组化分析结果, 并参照能谱仪的分析结果得知, 梭梭、白梭梭、沙拐枣和木本猪毛菜的叶片或同化枝中所含晶体的主要成分为草酸钙 。通过比较解剖结构发现, 梭梭和白梭梭的同化枝中含晶细胞最多, 其它3种植物的同化器官中含晶细胞较少, 而沙拐枣同化枝中有粘液细胞存 在。     

芋营养器官中的草酸钙晶体及其可能的生理作用

利用徒手切片,在光学显微镜下对芋(Colocasia esculenta(L.)Schott)营养器官中晶体的类型和分布进行了观察和研究,并用化学方法对晶体的化学成分进行了鉴定。结果表明,芋营养器官中的晶体为草酸钙结晶体,形态上可以分为针晶和簇晶两大类。含针晶束的异细胞有3种类型:含发射型草酸钙针晶束异细胞(存在于叶片、叶柄、块茎中),含大型草酸钙针晶束异细胞(存在于叶片、叶柄、块茎、块茎皮中),含大量草酸钙针晶的管状异细胞(仅存在于不定根中)。草酸钙针晶也有散乱分布于块茎和不定根中的。草酸钙簇晶在叶片、叶柄、块茎、块茎皮、不定根中均有分布,且叶片、叶柄、块茎皮中的簇晶比块茎和不定根中的尖锐。芋营养器官中的草酸钙晶体很可能是作为一种防御机制,防止动物的取食。     

The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med‐fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well‐known as invertebrate‐active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N‐terminal sequencing and by comparison with the genome sequence of strain 15.1. Synthesis of crystals by a plasmid‐cured derivative of strain 15.1 (produced using a novel curing strategy), demonstrated that the oxalate decarboxylase was encoded chromosomally. Crystals spontaneously solubilized when kept at low temperatures, and the protein produced was resistant to trypsin treatment. The insoluble crystals produced by B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae, but once the OxdD protein was solubilized, an increase of toxicity was observed. We also demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate decarboxylase is the first report of the natural production of parasporal inclusions of an enzyme.     

Subcellular distribution of calcium during spermatogenesis of zebrafish,Danio rerio

Calcium plays a variety of vital regulatory functions in many physiological and biochemical events in the cell. The aim of this study was to describe the ultrastructural distribution of calcium during different developmental stages of spermatogenesis in a model organism, the zebrafish (Danio rerio ), using a combined oxalate–pyroantimonate technique. Samples were treated by potassium oxalate and potassium pyroantimonate during two fixation stages and examined using transmission electron microscopy to detect electron dense intracellular calcium. The subcellular distribution of intracellular calcium was characterized in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. The area which is covered by intracellular calcium in different stages was quantified and compared using software. Isolated calcium deposits were mainly detectable in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. In the spermatid, calcium was partially localized in the cytoplasm as isolated deposits. However, most calcium was transformed from isolated deposits into an unbound pool (free calcium) within the nucleus of the spermatid and the spermatozoon. Interestingly, in the spermatozoon, calcium was mainly localized in a form of an unbound pool which was detectable as an electron‐dense mass within the nucleus. Also, sporadic calcium deposits were scattered in the midpiece and flagellum. The proportional area which was covered by intracellular calcium increased significantly from early to late stages of spermatogenesis. The extent of the area which was covered by intracellular calcium in the spermatozoon was the highest compared to earlier stages. Calcium deposits were also observed in the somatic cells (Sertoli, myoid, Leydig) of zebrafish testis. The notable changes in the distribution of intracellular calcium of germ cells during different developmental stages of zebrafish spermatogenesis suggest its different homeostasis and physiological functions during the process of male gamete development.     

Medicago truncatula-derived calcium oxalate crystals have a negative impact on chewing insect performance via their physical properties

Plant structural traits often act as defenses against herbivorous insects, causing them to avoid feeding on a given plant or tissue. Mineral crystals of calcium oxalate in Medicago truncatula Gaertn. (Fabaceae) leaves have previously been shown to be effective deterrents of lepidopteran insect feeding. They are also inhibitors of conversion of plant material into insect body mass during or after consumption. Growth of beet armyworm, Spodoptera exigua Hübner (Lepidoptera: Noctuidae), larvae was correspondingly greater on calcium oxalate‐defective (cod) mutants of M. truncatula with lower levels of crystal accumulation. Data presented here show that insects feeding on M. truncatula leaves with calcium oxalate crystals experience greater negative effects on growth and mandible wear than those feeding on artificial diet amended with smaller amorphous crystals from commercial preparations. Commercial calcium oxalate can be added to insect artificial diet at levels up to 7.5‐fold higher than levels found in wild‐type M. truncatula leaves with minimal effect on insect growth or lepidopteran mandibles. These data suggest that negative impacts of calcium oxalate in the diet of larvae are due to physical factors, and not toxicity of the compound, as high levels of the commercial crystals are readily tolerated. In contrast to the dramatic physical effects that M. truncatula‐derived crystals have on insect mandibles, we could detect no damage to insect peritrophic gut membranes due to consumption of these crystals. Taken together, the data indicate that the size and shape of prismatic M. truncatula oxalate crystals are important factors in determining effects on insect growth. If manipulation of calcium oxalate is to be used in developing improved insect resistance in plants, then our findings suggest that controlling not only the overall amount, but also the size and shape of crystals, could be valuable traits in selecting desirable plant lines.     

Cells with Crystals of Calcium Oxalate in the Leaves of Phaseolus vulgaris — a Comparison with those in Canavalia ensiformis

Leaflets of Phaseolus vulgaris contain crystals of calcium oxalate in the adaxial bundle sheath extensions. Most of the crystals accompany the lateral veins of third order. The average oxalate content of the leaves is 0.8% of dry weight. Some features of leaflet anatomy of Phaseolus and Canavalia are shown and the possible relation of anatomy to localization and development of crystals in each of the species is discussed. The majority of crystals in Phaseolus originate with the young leaflets newly unfolded. Calcium deficiency reduces number and size of crystals.     

Carbonate Accumulation in the Bark of Terminalia bellirica: A New Habitat for the Oxalate-Carbonate Pathway

Oxalate oxidation and carbonate precipitation associated with the oxalogenic tree Terminalia bellirica were investigated. Calcium oxalate crystals, oxalotrophic bacteria (dominated by genera Methylobacterium and Burkholderia), and carbonate accumulation (82% dry weight), were detected in the bark. In contrast, only a slight accumulation of carbonate was observed in soil (1.5%). The combined geochemical and microbiological analyses suggest that bark of an Indian living tree represents a novel habitat for the accumulation of carbonate associated with bacterial oxalate oxidation. The importance of these types of habitats needs to be quantified when considering the biogeochemical cycling of carbon and calcium in tropical ecosystems.     

Insolubilization of potassium chloride crystals in Tradescantia pallida

Summary. Calcium oxalate crystals are by far the most prevalent and widely distributed mineral deposits in higher plants. In Tradescantia pallida, an evergreen perennial plant widely used as an ornamental plant, calcium oxalate crystals occur in the parenchymal tissues of stem, leaf, and root, as well as in flower organs, in the form of either raphides or tetragonal prismatic crystals or both. Energy-dispersive X-ray analysis revealed that C, O, and Ca were the main elements; and K, Cl, and Si, the minor elements. Infrared and X-ray analyses of crystals collected from these tissues detected the coexistence of two calcium oxalate chemical forms, i.e., whewellite and weddellite, as well as calcite, opal, and sylvite. Here, we show for the first time the occurrence of epitaxy in mineral crystals of plants. Epitaxy, which involves the oriented overgrowth of one crystal onto a second crystalline substrate, might explain how potassium chloride (sylvite) – one of the most water-soluble salts – stays insoluble in crystal form when coated with a calcium oxalate epilayer. The results indicate the potential role of crystals in regulating the ionic equilibrium of both calcium and potassium ions. Correspondence and reprints: Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EGA, Ciudad de Buenos Aires, Argentina.     

A study of calcium binding and uptake by isolated cardiac sarcoplasmic reticulum: the use of a new ionophore (X537A)

Calcium “binding” (absence of oxalate) and “uptake” (presence of oxalate) were studied in isolated cardiac sarcoplasmic reticulum. X537A (< 5 μg/mg protein) inhibits binding and uptake similarly; the same concentrations induce a highly significant augmented calcium release from binding sites only in the absence of oxalate. At higher concentrations, “uptake” is virtually eliminated while “binding” (I₅₀ = 16 μg/mg) is less inhibited suggesting an additional action of X537A on a step unique to uptake. Data suggest that “binding” and “transport” of calcium may be different but they may initially share sites.     

Heavy metal stress reduces the deposition of calcium oxalate crystals in leaves of Phaseolus vulgaris

Calcium oxalate (CaOx) crystals in plants may serve as a sink for the absorption of excess calcium, and they could play an important role in heavy metal detoxification. In this study, the effect of heavy metals and different calcium concentrations on the growth of calcium oxalate crystals in leaves of Phaseolus vulgaris was investigated. Different analytical techniques were used to determine the influence of exogenous lead and zinc on CaOx deposition and to detect a presence of these metals in CaOx crystals. We found a positive correlation between the calcium concentration in the nutrient medium and the production of calcium oxalate crystals in leaves of hydroponically grown plants. On the other hand, addition of the heavy metals to the nutrient medium decreased the number of crystals. Energy dispersive X-ray spectrometry did not detect the inclusion of heavy metals inside the CaOx crystals. Our investigation suggests that CaOx crystals do not play a major role in heavy metal detoxification in P. vulgaris but do play an important role in bulk calcium regulation.     

Calcium fertilization increases the concentration of calcium in sapwood and calcium oxalate in foliage of red spruce

Calcium cycling plays a key role in the health and productivity of red spruce forests in the northeastern US. A portion of the flowpath of calcium within forests includes translocation as Ca²⁺ in sapwood and accumulation as crystals of calcium oxalate in foliage. Concentrations of Ca in these tree tissues have been used as markers of environmental change due to acidic deposition or forest management practices. We compared the effects of Ca fertilization treatment on Ca concentration in wood and Ca and oxalate (Ox) concentration in foliage at two locations with different initial concentrations of Ca in the soil. We found greater amounts of Ca in wood from the high-Ca location than from the low-Ca location. Ca concentration was greater in wood formed in the 1970s than for wood formed in the 1980s, the outermost decadal band in these samples. The Ca-treatment was detected as an increased concentration of Ca in the 1970s and 1980s decadal bands. We also found that variation in Ca and Ox in foliage was essentially stoichiometric. The appearance and response to chemical tests of crystals in foliage were consistent with identification as calcium oxalate. The increased Ca in wood after Ca-treatment of the soil supports the use of dendrochemistry of base cations to investigate environmental change. However, differences in Ca concentration between the two outermost decadal bands of wood illustrate that internal processes of translocation and storage also affect Ca concentration. Calcium oxalate production in foliage diverts carbon from ordinary biosynthesis and energy-yielding processes. This sequestration, shedding, and decomposition of foliage may represent a significant and under-recognized contribution to carbon and Ca cycling.     

The Isolation and Properties of Oxalate Crystals from Plants

A method of isolation of crystalline inclusions of plant cellsis described. The crystals consist mainly of calcium oxalatein plants grown under normal conditions, but when calcium isreplaced by magnesium, barium, or strontium in the culture solutionthese elements substitute for calcium in the crystals; evenunder normal conditions magnesium occurs in the crystals tothe extent of about 2 per cent. The crystal morphology vanesin the species examined from raphides to complex conglomeratesand X-ray diffraction demonstrates an association of raphideswith calcium oxalate monohydrate whilst other solitary formsand conglomerates are associated with calcium oxalate 2.25H₂O.On this basis the species examined can be divided mto threegroups.     

Developmental features of calcium oxalate crystal sand deposition inBeta vulgaris L. Leaves

Summary Sugar beet (Beta vulgaris L.) leaf has a layer of cells extended laterally between the palisade parenchyma and spongy mesophyll that develop numerous small crystals (crystal sand) within their vacuoles. Solubility studies and histochemical staining indicate the crystals are calcium oxalate. The crystals are deposited within the vacuoles early during leaf development, and at maturity the cells are roughly spherical in shape and 2 to 3 times larger than other mesophyll cells. Crystal deposition is preceeded by formation of membrane vesicles within the vacuole. The membranes are synthesizedde novo in the vacuole and have a typical trilaminate structure as viewed with the TEM. The membranes are formed within paracrystalline aggregates of tubular particles (6–8nm outer diameter) as membrane sheets, but are later organized into chambers or vesicles. Calcium oxalate is then precipitated within the membrane chambers. The tubular particles involved in membrane synthesis are usually present in the vacuoles of mature crystal cells, but in very small amounts.