Crystal morphology and carbon/carbon composition of solid oxalate in cacti

Morphology, crystal structure, and carbon isotopic composition of calcium oxalate from representative species from the family Cactaceae were determined using scanning electron microscopy, x-ray diffraction, and isotope ratio mass spectrometry. Crystals from one species in the Opuntieae tribe of the Cactaceae were druses with acute points composed of the monohydrate form of calcium oxalate (whewellite). Crystals from three species in the Cereeae tribe were the dihydrate form of calcium oxalate (weddellite) forming druses made up of tetragonal and isodiametric crystallites. The oxalate was relatively enriched in ¹³C isotope (-7.3 to - 8.7 ‰) compared with woody fibers (-13.3 to 14.1 ‰) from the same plants.     

Complex biomineralization pattern in cactaceae

The infrared spectroscopic investigation of biominerals isolated from different Cactaceae species belonging to the Opuntioideae subfamily shows the presence of a very complex mineral composition, including whewellite (monohydrated calcium oxalate), opal (SiO2) and calcite (CaCO3). This is the first report on the presence of a calcium carbonate in these types of plants.     

Trichoderma koningii as a biomineralizing fungous agent of calcium oxalate crystals in typical Argiudolls of the Los Padres Lake natural reserve (Buenos Aires, Argentina)

The aim of the present study, performed on typical Argiudolls in a natural reserve with little or no anthropic impact, was to characterize the fungous biomineralizing process of calcium oxalate crystals in organic horizons of the soil. The chosen sites possessed different plant cover, identified as acacia woods and grassy meadows with particular micro environmental conditions that have differing effects in the process of biomineralization. The contribution of the plant material in the soil is a key factor since 1) it generates the particular composition of the organic horizons, 2) it determines the nature of decomposing organisms, and 3) it affects the presence, composition and development of biominerals. According to the results obtained, the acacia woods prove to be a site comparatively more favorable to the fungous biomineralizing process. This makes itself manifest in the greater abundance and development of crystals in the organic horizons of the soil, resulting in whewellite (CaC2O4.H2O) and weddellite (CaC2O4.(2+x) H2O) regarding biomineral species developed, the latter being the major component. The observation of both species of biominerals is noteworthy since it represents the first cited in the country. The isolated fungous organisms were Trichoderma koningii, and Absidia corymbifera. T. koningii was identified as the most active biomineralizing organism thus constituting the first reference to indicate this species as a biomineral producing agent.     

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.     

Calcium oxalate and sulphate-containing structures on the thallial surface of the lichen Ramalina lacera: response to polluted air and simulated acid rain

The formation of calcium‐containing structures on the thallial surface of the lichen Ramalina lacera (With.) J.R. Laund. in response to air pollution and to simulated acid rain, was studied in in situ and transplanted thalli. In situ thalli were collected from an unpolluted site and transplanted to heavily polluted and less polluted sites for a 10 month period. Additional thalli were treated either with double distilled water or with simulated acid rain. Scanning electron microscopy and infrared spectrometry revealed that thallial surfaces of in situ R. lacera samples collected in unpolluted sites were covered with two kinds of calcium oxalate crystals: whewellite and weddellite. These aggregates of calcium oxalate crystals appear to disintegrate and provide a crystal layer on the thallial surface. Infrared spectroscopy of powder scraped from thallial surfaces of transplants, retrieved from non‐polluted sites, showed the presence of whewellite and weddellite, whereas powders obtained from thalli retrieved from polluted sites contained whewellite, weddellite and gypsum. It is suggested that a certain fraction of the gypsum detected in crater‐like structures in transplants from polluted sites and in thalli treated with simulated acid rain is endogenous and should be considered a biomineral.     

Experimental calcium-oxalate crystal production and dissolution by selected wood-rot fungi

Twenty-six species of white-rotting Agaricomycotina fungi (Basidiomycota) were screened for their ability to produce calcium-oxalate (CaOx) crystals in vitro. Most were able to produce CaOx crystals in malt agar medium in the absence of additional calcium. In the same medium enriched with Ca²⁺, all the species produced CaOx crystals (weddellite or whewellite). Hyphae of four species (Ganoderma lucidum, Polyporus ciliatus, Pycnoporus cinnabarinus, and Trametes versicolor) were found coated with crystals (weddellite/whewellite). The production of CaOx crystals during the growth phase was confirmed by an investigation of the production kinetics for six of the species considered in the initial screening (Pleurotus citrinopileatus, Pleurotus eryngii, Pleurotus ostreatus, P. cinnabarinus, Trametes suaveolens, and T. versicolor). However, the crystals produced during the growth phase disappeared from the medium over time in four of the six species (P. citrinopileatus, P. eryngii, P. cinnabarinus, and T. suaveolens). For P. cinnabarinus, the disappearance of the crystals was correlated with a decrease in the total oxalate concentration measured in the medium from 0.65 ??g mm⁻² (at the maximum accumulation rate) to 0.30 ??g mm⁻². The decrease in the CaOx concentration was correlated with a change in mycelia morphology. The oxalate dissolution capability of all the species was also tested in a medium containing calcium oxalate as the sole source of carbon (modified Schlegel medium). Three species (Agaricus blazei, Pleurotus tuberregium, and P. ciliatus) presented a dissolution halo around the growth zone. This study shows that CaOx crystal production is a widespread phenomenon in white-rot fungi, and that an excess of Ca²⁺ can enhance CaOx crystal production. In addition, it shows that some white-rot fungal species are capable of dissolving CaOx crystals after growth has ceased. These results highlight a diversity of responses around the production or dissolution of calcium oxalate in white-rot fungi and reveal an unexpected potential importance of fungi on the oxalate cycle in the environment.     

Evidence of formation of glushinskite as a biomineral in a Cactaceae species

The X-ray diffractometric and infrared spectroscopic investigation of crystalline material isolated from the Cactaceae species Opuntia ellisiana shows the presence of a very complex mineral composition, including whewellite (monohydrated calcium oxalate), opal (SiO2), calcite (CaCO3) and glushinskite (dihydrated magnesium oxalate). This is the first report of the presence of magnesium oxalate in plants.     

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.     

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.     

虎掌（天南星科）中草酸钙晶体的形态和分布

在光学显微镜下对虎掌（Pinellia pedatisecta）营养器官和繁殖器官中晶体的类型和分布进行了观察和分析,探讨晶体的功能与作用机制。结果表明：（1）虎掌各个器官中都发现有晶体,且晶体类型有针晶、簇晶、砂晶和柱晶4种形态,其中针晶最为常见。（2）虎掌叶中的晶体大多以针晶状分布在叶片上表皮下的叶肉中,少数分布在叶下表皮下的叶肉中,其次砂晶和星芒状簇晶也在叶中较常见,叶中也有少量的柱晶。（3）虎掌的块茎中分布有大量的针晶束,在输导组织附近还有一些大的簇晶;虎掌的不定根中分布有不整齐的针晶和排列不规则的针晶束以及少量大的簇晶。（4）虎掌的佛焰苞中分布有针晶、簇晶和砂晶,且在佛焰苞中的针晶主要分布于上、下表皮之下的叶肉中,砂晶多分布在佛焰苞的上、下表皮上。（5）虎掌的花药壁中分布有针晶束,其方向和花药壁表面垂直,而花粉囊中只有小的簇晶。（6）虎掌的果皮和种皮上分布有大量的针晶。根据晶体在酸中的溶解性,虎掌体内所有晶体的化学成分都为草酸钙。研究认为,虎掌各个器官中的各种草酸钙晶体对于保护虎掌免受食草动物取食具有重要的作用。     

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 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     

Identification,distribution, and quantification of biominerals in a deciduous forest

Biomineralization is a common process in most vascular plants, but poorly investigated for trees. Although the presence of calcium oxalate and silica accumulation has been reported for some tree species, the chemical composition, abundance, and quantification of biominerals remain poorly documented. However, biominerals may play important physiological and structural roles in trees, especially in forest ecosystems, which are characterized by nutrient‐poor soils. In this context, our study aimed at investigating the morphology, distribution, and relative abundance of biominerals in the different vegetative compartments (foliage, branch, trunk, and root) of Fagus sylvatica L. and Acer pseudoplatanus L. using a combination of scanning electron microscopy and tomography analyses. Biomineral crystallochemistry was assessed by X‐ray diffraction and energy‐dispersive X‐ray analyses, while calcium, silicon, and oxalic acid were quantified in the compartments and at the forest scale. Our analyses revealed that biominerals occurred as crystals or coating layers mostly in bark and leaves and were identified as opal, whewellite, and complex biominerals. In both tree species, opal was mostly found in the external tissues of trunk, branch, and leaves, but also in the roots of beech. In the stand, opal represents around 170 kg/ha. Whewellite was found to suit to conductive tissues (i.e., axial phloem parenchyma, vascular bundles, vessel element) in all investigated compartments of the two tree species. The shape of whewellite was prismatic and druses in beech, and almost all described shapes were seen in sycamore maple. Notably, the amount of whewellite was strongly correlated with the total calcium in all investigated compartments whatever the tree species is, suggesting a biologic control of whewellite precipitation. The amount of whewellite in the aboveground biomass of Montiers forest was more important than that of opal and was around 1170 kg/ha. Therefore, biominerals contribute in a substantial way to the biogeochemical cycles of silicon and calcium.     

Crystals in calyces of Lamiaceae and their phylogenetic and adaptive significance

The diversity of crystal types is studied in fruiting calyces of 435 out of the c. 7,200 species, and 184 out of the c. 236 genera of Lamiaceae, mainly in the subfamilies Nepetoideae and Lamioideae. Calcium oxalate crystals are found in about half of the studied calyces, and belong to the following main types: prismatics in the inner epidermis, prismatics in fibres, prismatics in sclereids, prismatics in the mesophyll and druses in the mesophyll. Presence of epidermal prismatics may constitute a synapomorphy of the subfamily Nepetoideae or of a subclade within this subfamily consisting of the two largest tribes Mentheae and Ocimeae. The presence of narrow mesophyll prismatics seems to support a large clade of Lamioideae genera in a recent molecular phylogeny of the subfamily. Epidermal prismatics are found to be particularly frequent in closed calyces, calyces with a narrow mouth and internally hairy calyces. These findings and the position of the crystals in the calyces are regarded as supporting the hypothesis that calcium oxalate crystals protect the fruit against insect predators.     

First evidence for the presence of weddellite crystallites in Opuntia ficus indica parenchyma

Calcium oxalate crystallites occur very often in the plants tissues and their role is still poorly known. We report here the experimental protocol leading to the isolation of two forms of calcium oxalate crystallites differing in their hydration level in the parenchymal tissues of Opuntia ficus indica (Miller). Whereas the whewellite crystallites are habitual in all Opuntia species, the weddellite form has never been isolated from these species before, which is probably due to their small size (about 1 microm). We have identified these forms using X-ray diffraction and scanning electron microscopy.     

Characterization of biominerals in species of Canna (Cannaceae)

Plant biominerals are not always well characterized, although this information is important for plant physiology and can be useful for taxonomic purposes. In this work, fresh plant material of seven wild neotropical species of genus Canna, C. ascendens, C. coccinea, C. indica, C. glauca, C. plurituberosa, C. variegatifolia and C. fuchsina sp. ined., taken from different habitats, were studied to characterize the biominerals in their internal tissues. For the first time, samples from primary and secondary veins of leaves were investigated by means of infrared spectroscopy, complemented with X-ray powder diffractometry and scanning electron microscopy. The spectroscopic results, supported by X-ray powder diffractometry, suggest that the calcium oxalate is present in the form of whewellite (CaC2O4 x H2O) in all the investigated samples. It is interesting to emphasize that all IR spectra obtained were strongly similar in all species studied, thus indicating an identical chemical composition in terms of the biominerals found. In this sense, the results suggest that the species of Canna show similar ability to produce biogenic silica and produce an identical type of calcium oxalate within their tissues. These results can be an additional trait to support the relationship among the families of Zingiberales.     

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

The morphology and distribution of intracellular crystals of calcium oxalate in taro (Colocasia esculenta) was studied by light microscopy. The modified Pizzolato (AgNO₃-H₂O₂) method was used to localize crystals in cleared corm cross sections. Crystals of two forms were found: druses and raphides. The numbers and density of the crystals in corms increase rapidly in early development, then level off, and eventually decrease in older and larger corms. An especially high concentration of druses was observed 2-3 mm from the exterior edge of many corms. This corresponds to a ring of vascular tissue which circumscribes the corm at approximately the same distance from the surface. Observations suggest that the development of these highly specialized cells and the formation of calcium oxalate crystals is a dynamic process.     

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.     

Crystallochemical characterization of calcium oxalate crystals isolated from seed coats of Phaseolus vulgaris and leaves of Vitis vinifera

Calcium oxalate crystals are a major biomineralization product in higher plants. Their biological function and use are not well understood. In this work, we focus on the isolation and crystallochemical characterization of calcium oxalate crystals from seed coats of Phaseolus vulgaris (prisms) and leaves of Vitis vinifera (raphides and druses) using ultrastructural methods. A proposal based on crystal growth theory was used for explaining the existence of different morphologies shown by these crystals grown inside specialized cells in plants.