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.     

Characterization of calcium oxalates generated as biominerals in cacti

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

Mucilage Cells, Calcium Oxalate Crystals and Soluble Calcium in Opuntia ficus-indica

The subcellular location of soluble calcium in parenchymatousand mucilage cells of Opuntia ficus-indica was determined histochemically.Soluble calcium was observed in crystal chambers containingcalcium oxalate on the membrane of the vesicles. Calcium wasalso present in the plasmalemma, in plasmodesmata, in cell walls,in mitochondria and in the vacuoles. Especially marked was thepresence of soluble calcium in vesicles free or fused with theplasmalemma. Little free calcium was observed in other cellcompartments. In the calcium economy of tissues the location of soluble calciumand the transport of calcium to and from mucilage cells to parenchymatouscells and calcium oxalate idioblasts will play a role. Chelationof calcium by mucilage or oxalate, which depends on pH, ionicstrength, etc., will be important in this respect. Opuntia ficus-indica, calcium oxalate, mucilage cells, transport of calcium     

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.     

Traditional knowledge and genetic diversity ofopuntia pilifera (cactaceae) in the tehuacán-cuicatlán valley, Mexico

Traditional Knowledge and Genetic Diversity of Opuntia pilifera (Cactaceae) in the Tehuacán-Cuicatlán Valley, Mexico. Economic Botany 59(4)366-376, 2005. Most studies of the genusOpuntia have focused on economically important species, and therefore more knowledge concerning the genetic diversity among wild and locally managedOpuntia species is needed for an expanded use of cacti in the future. The present study is part of ongoing ethnobotanical work in the Tehuacán-Cuicatlán Valley of Mexico and focuses on six traditionally classified forms ofOpuntia pilifera used as food by the indigenous Popoloca people in San Juan Atzingo. Traditional knowledge of how to distinguish these forms based on fruit flavor, color, size, and number of spines on the fruits and cladodes is preserved in the local community. Genetic fingerprinting with 129 AFLPs did not correlate with this traditional morphological classification of 67 cacti. Yet, these AFLPs distinguished the analyzed 67Opuntia pilifera cacti easily from the out-group comprising 17 wildOpuntia velutina.     

Phylogeny of Opuntia s.s. (Cactaceae): Clade delineation, geographic origins, and reticulate evolution

? Premise of the study: The opuntias (nopales, prickly pears) are not only culturally, ecologically, economically, and medicinally important, but are renowned for their taxonomic difficulty due to interspecific hybridization, polyploidy, and morphological variability. Evolutionary relationships in these stem succulents have been insufficiently studied; thus, delimitation of Opuntia s.s. and major subclades, as well as the biogeographic history of this enigmatic group, remain unresolved. ? Methods: We sequenced the plastid intergenic spacers atpB-rbcL, ndhF-rpl32, psbJ-petA, and trnL-trnF, the plastid genes matK and ycf1, the nuclear gene ppc, and ITS to reconstruct the phylogeny of tribe Opuntieae, including Opuntia s.s. We used phylogenetic hypotheses to infer the biogeographic history, divergence times, and potential reticulate evolution of Opuntieae. ? Key results: Within Opuntieae, a clade of Tacinga, Opuntia lilae, Brasiliopuntia, and O. schickendantzii is sister to a well-supported Opuntia s.s., which includes Nopalea. Opuntia s.s. originated in southwestern South America (SA) and then expanded to the Central Andean Valleys and the desert region of western North America (NA). Two major clades evolved in NA, which subsequently diversified into eight subclades. These expanded north to Canada and south to Central America and the Caribbean, eventually returning back to SA primarily via allopolyploid taxa. Dating approaches suggest that most of the major subclades in Opuntia s.s. originated during the Pliocene. ? Conclusions: Opuntia s.s. is a well-supported clade that includes Nopalea. The clade originated in southwestern SA, but the NA radiation was the most extensive, resulting in broad morphological diversity and frequent species formation through reticulate evolution and polyploidy.     

Host-plant shifts and adult survival in the cactus breeding Drosophila mojavensis

Abstract. 1. In order to gain insight into how Drosophila mojavensis Patterson and Crow (which lives on the rotting tissue of cactus) successfully shifted host plants, from an Opuntia species to columnar cactus species, over evolutionary time, specific components of columnar cactus and Optunia tussue were examined for their effects on adult longevity.
2. The availability of free sugars affects the survival of adult D.mojavensis . The addition of a sugar to columnar cactus tissue significantly increased the longevity of adults that fed on those tissues. This increase in adult longevity was equal to the longevity observed on unsupplemented Opuntia tissue, which has free sugars present.
3. Live microorganisms added to Opuntia tissue had a slight detrimental effect on adult longevity (longevity was decreased by approximately 1 week).
4. Low concentrations of atmospheric ethanol (independent of diet) increased the longevity of adult D.mojavensis to levels comparable in magnitude to that provided by the addition of free sugars to the diet.
5. This information and the apparent lack of a year-round supply of free sugars for D.mojavensis in nature suggests that the ethanol (produced by fermentation) rather than free sugars per se is the most important component of rotting cactus tissue for the survival of this species and may explain its successful host-plant shift.     

Oviposition acceptance and fecundity schedule in the cactophilic sibling species Drosophila buzzatii and D. koepferae on their natural hosts

We tested for the occurrence of oviposition acceptance for different media prepared with cactus tissues of three alternative cactus hosts: Opuntia sulphurea, O. quimilo and Trichocereus terschekii for 4 consecutive days in lines of two Drosophila buzzatii populations and one population of D. koepferae. Our results showed that the former laid significantly more eggs on both Opuntia cacti than on T. terschekii, whereas D. koepferae preferred T. terschekii. In addition, fecundity schedules differed between species: D. buzzatii laid similar numbers of eggs along the four-day sampling period, whereas D. koepferae showed an oviposition peak on the second day of egg collection on T. terschekii. We suggest that the between-species disparities observed in oviposition acceptance and fecundity schedule may be related to the temporal and spatial predictability of Opuntia versus T. terschekii (cardón) as part of the different adaptive strategies that have evolved after the split of D. koepferae and D. buzzatii from their recent common ancestor. Therefore, the willingness to accept hosts would be an important factor in the habitat selection and in the maintenance of species diversity.     

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     

Biominerals and waxes of <Emphasis Type="Italic">Calamagrostis epigejos</Emphasis> and <Emphasis Type="Italic">Phragmites australis</Emphasis> leaves from post-industrial habitats

Vascular plants are able to conduct biomineralization processes and collect synthesized compounds in their internal tissues or to deposit them on their epidermal surfaces. This mechanism protects the plant from fluctuations of nutrient levels caused by different levels of supply and demand for them. The biominerals reflect both the metabolic characteristics of a vascular plant species and the environmental conditions of the plant habitat. The SEM/EDX method was used to examine the surface and cross-sections of the Calamagrostis epigejos and Phragmites australis leaves from post-industrial habitats (coal and zinc spoil heaps). The results from this study have showed the presence of mineral objects on the surfaces of leaves of both grass species. The calcium oxalate crystals, amorphous calcium carbonate spheres, and different silica forms were also found in the inner tissues. The high variety of mineral forms in the individual plants of both species was shown. The waxes observed on the leaves of the studied plants might be the initializing factor for the crystalline forms and structures that are present. For the first time, wide range of crystal forms is presented for C. epigejos. The leaf samples of P. australis from the post-industrial areas showed an increased amount of mineral forms with the presence of sulfur.     

Selenium accumulation, distribution, and speciation in spineless prickly pear cactus: a drought- and salt-tolerant, selenium-enriched nutraceutical fruit crop for biofortified foods

The organ-specific accumulation, spatial distribution, and chemical speciation of selenium (Se) were previously unknown for any species of cactus. We investigated Se in Opuntia ficus-indica using inductively coupled plasma mass spectrometry, microfocused x-ray fluorescence elemental and chemical mapping (μXRF), Se K-edge x-ray absorption near-edge structure (XANES) spectroscopy, and liquid chromatography-mass spectrometry (LC-MS). μXRF showed Se concentrated inside small conic, vestigial leaves (cladode tips), the cladode vasculature, and the seed embryos. Se K-edge XANES demonstrated that approximately 96% of total Se in cladode, fruit juice, fruit pulp, and seed is carbon-Se-carbon (C-Se-C). Micro and bulk XANES analysis showed that cladode tips contained both selenate and C-Se-C forms. Inductively coupled plasma mass spectrometry quantification of Se in high-performance liquid chromatography fractions followed by LC-MS structural identification showed selenocystathionine-to-selenomethionine (SeMet) ratios of 75:25, 71:29, and 32:68, respectively in cladode, fruit, and seed. Enzymatic digestions and subsequent analysis confirmed that Se was mainly present in a "free" nonproteinaceous form inside cladode and fruit, while in the seed, Se was incorporated into proteins associated with lipids. μXRF chemical mapping illuminated the specific location of Se reduction and assimilation from selenate accumulated in the cladode tips into the two LC-MS-identified C-Se-C forms before they were transported into the cladode mesophyll. We conclude that Opuntia is a secondary Se-accumulating plant whose fruit and cladode contain mostly free selenocystathionine and SeMet, while seeds contain mainly SeMet in protein. When eaten, the organic Se forms in Opuntia fruit, cladode, and seed may improve health, increase Se mineral nutrition, and help prevent multiple human cancers.     

Elucidation of multiple forms of nephrocalcin by P-NMR spectrometer

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

Biologically induced mineralization in the tree Milicia excelsa (Moraceae): its causes and consequences to the environment

Iroko trees (Milicia excelsa) in Ivory Coast and Cameroon are unusual because of their highly biomineralized tissues, which can virtually transform the trunk into stone. Oxalic acid (C₂O₄H₂) and metal‐oxalate play important roles in their ecosystems. In this study, the various forms of oxalate and carbonate mineralization reactions are investigated by using scanning electron microscopy and X‐ray diffraction. Calcium oxalate monohydrate is associated with stem, bark and root tissues, whereas calcium oxalate dihydrate is found with wood rot fungi in soils, as well as in decaying wood. Laboratory cultures show that many soil bacteria are able to oxidize calcium oxalate rapidly, resulting in an increase in solution pH. In terms of M. excelsa, these transformations lead to the precipitation of calcium carbonate, not only within the wood tissue, but also within the litter and soil. We calculate that c. 500 kg of inorganic carbon is accumulated inside an 80‐year‐old tree, and c. 1000 kg is associated with its surrounding soil. Crucially, the fixation of atmospheric CO₂ during tree photosynthesis, and its ultimate transformation into calcite, potentially represents a long‐term carbon sink, because inorganic carbon has a longer residence time than organic carbon. Considering that calcium oxalate biosynthesis is widespread in the plant and fungal kingdoms, the biomineralization displayed by M. excelsa may be an extremely common phenomena.     

The role of a spiny plant refuge in structuring grazed shortgrass steppe plant communities

This study evaluates the hypothesis that biological grazing refuges have an important role in plant-grazer interactions of grasslands with a long history of grazing. We assessed the hypothesis that clumps of the spiny cactus Opuntia polyacantha provide biological refuges from cattle grazing, affecting cover and seedhead production of associated vascular plants in the shortgrass steppe of the North America. The study was based on sampling inside and outside Opuntia clumps in eight long-term moderately grazed pastures established 60 yr ago and their respective ungrazed controls. Opuntia clumps provided a refuge for seedhead production of the dominant grass ( Bouteloua gracilis ) and for cover and seedhead production of many plant groups. Clumps were also a refuge for species sensitive to grazing (species that decrease with grazing) and barrel-cacti, but not for species preferred by cattle (species with greater proportion in the diet than in the field), exotics or weeds. Our results suggest that these effects were mainly through changes in the microenvironmental conditions resulting from protection effects, even though all potential microenvironmental effects could not be measured. Cacti promoted some negative effects on other plant groups, probably due to the space occupied by cladodes inside cactus clumps. The refuge effects observed at the group level did not translate into strong community level effects. Species diversity ( H' ) was greater in cactus clumps due to lower dominance rather than greater richness. The presence of Opuntia clumps increased landscape-scale diversity. This ecological role of Opuntia clumps as refuge from cattle grazing should be taken into consideration in management practices aimed at cactus eradication in order to increase forage availability for livestock. We discuss the potential role of plant community productivity and grazing history with regard to the importance of natural refuges in structuring grassland communities.     

Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny of the Drosophila repleta species group

We present a revised molecular phylogeny of the Drosophila repleta group including 62 repleta group taxa and nine outgroup species based on four mitochondrial and six nuclear DNA sequence fragments. With ca. 100 species endemic to the New World, the repleta species group represents one of the major species radiations in the genus Drosophila. Most repleta group species are associated with cacti in arid or semiarid regions. Contrary to previous results, maximum likelihood and Bayesian phylogenies of the 10-gene dataset strongly support the monophyly of the repleta group. Several previously described subdivisions in the group were also recovered, despite poorly resolved relationships between these clades. Divergence time estimates suggested that the repleta group split from its sister group about 21millionyears ago (Mya), although diversification of the crown group began ca. 16Mya. Character mapping of patterns of host plant use showed that flat leaf Opuntia use is common throughout the phylogeny and that shifts in host use from Opuntia to the more chemically complex columnar cacti occurred several times independently during the history of this group. Although some species retained the use of Opuntia after acquiring the use of columnar cacti, there were multiple, phylogenetically independent instances of columnar cactus specialization with loss of Opuntia as a host. Concordant with our proposed timing of host use shifts, these dates are consistent with the suggested times when the Opuntioideae originated in South America. We discuss the generally accepted South American origin of the repleta group.     

Developmental Time and Thorax Length Differences Between the Cactophilic Species Drosophila Buzzatii and D. Koepferae Reared in Different Natural Hosts

Drosophila buzzatii and Drosophila koepferae are two cactophilic sibling species whose ranges partially overlap in Northwestern and Western Argentina. Both species can utilize the decaying tissues of both Opuntia and columnar cacti as breeding sites. Though D. buzzatii and D. koepferae are not differentially attracted to Opuntia and columnar hosts, the composition of the communities of flies emerging from natural substrates of both cacti differed significantly in a natural population. The objective of this paper is to analyze whether intra and/or interspecific competition affects development time and thorax length in D. buzzatii and D. koepferae when both species are reared in single and mixed species culture and fed with semi-natural media prepared with fermenting materials of Opuntia sulphurea(tuna) and Trichocereus terschekii(cardón). Our results showed that both traits differ significantly between flies raised in different hosts and that differences between D. koepferae and D. buzzatii species for both thorax length and development time depend on the type of culture (mixed vs. single species). In addition, the host by type of culture interaction was significant. We also observed thorax length differences between Drosophila species and type of culture. Our present data suggest that the effect of intra and interspecific competition varied between the two traits investigated and between species. However, competition alone cannot explain the differential pattern of resource utilization shown by D. buzzatii and D. koepferae in the natural population studied.     

Calcium Oxalate Crystals in Eucalypt Ectomycorrhizae: Morphochemical Characterization

Ectomycorrhizal fungi are ubiquitous in forest ecosystems, benefitting plants principally by increasing the uptake of water and nutrients such as calcium from the soil. Previous work has demonstrated accumulation of crystallites in eucalypt ectomycorrhizas, but detailed morphological and chemical characterization of these crystals has not been performed. In this work, cross sections of acetic acid-treated and cleared ectomycorrhizal fragments were visualized by polarized light microscopy to evaluate the location of crystals within cortical root cells. Ectomycorrhizal sections were also observed by scanning electron microscopy (SEM) coupled with energy dispersive x-ray (EDS) microprobe analysis. The predominant forms of crystals were crystal sand (granules) and concretions. Calcium, carbon and oxygen were detected by EDS as constituent elements and similar elemental profiles were observed between both crystal morphologies. All analyzed crystalline structures were characterized as calcium oxalate crystals. This is the first report of the stoichiometry and morphology of crystals occurring in eucalypt ectomycorrhizas in tropical soils. The data corroborates the role of ectomycorrhizae in the uptake and accumulation of calcium in the form of calcium oxalate crystals in hybrid eucalypt plants.     

The effect of ultraviolet-depleted light on the flavonol contents of the cactus species Opuntia wilcoxii and Opuntia violacea

An early investigation at the Biosphere-2 Laboratory, an artificial ecosystem in the Arizona desert, had shown that the flavonoid content of cacti grown in glass-filtered solar light was lower than of cacti grown in normal solar light. This was attributed to the absence of ultraviolet (UV) radiation, which is required for flavonoid biosynthesis. In this study, two species of Opuntia cacti were grown in solar and UV-depleted light, and their flavonol contents of different tissues were determined by HPLC. O. wilcoxii, previously raised in the absence of UV light, was exposed to normal solar light. The flavonol content of young O. wilcoxii pads was 28-fold higher when grown in solar light as compared to UV-depleted light. The flavonol contents of mature outer tissues were only slightly higher. O. violacea, previously raised in solar light, was also maintained in the same UV-depleted artificial ecosystem. The flavonol content after hydrolysis of outer tissues was similar, whether grown in solar light or UV-depleted light. We attribute these responses to different biosynthetic and metabolic rates of young vs. mature plant tissues; slow-growing mature tissues neither produce nor metabolize compounds as quickly as immature tissues. These findings indicate that artificial ecosystems can influence the production of natural products in cultivated plants.     

Precipitation of calcium, magnesium, strontium and barium in tissues of four acacia species (leguminosae: mimosoideae)

Precipitation of calcium in plants is common. There are abundant studies on the uptake and content of magnesium, strontium and barium, which have similar chemical properties to calcium, in comparison with those of calcium in plants, but studies on co-precipitation of these elements with calcium in plants are rare. In this study, we compared morphologies, distributional patterns, and elemental compositions of crystals in tissues of four Acacia species grown in the field as well as in the glasshouse. A comparison was also made of field-grown plants and glasshouse-grown plants, and of phyllodes of different ages for each species. Crystals of various morphologies and distributional patterns were observed in the four Acacia species studied. Magnesium, strontium and barium were precipitated together with calcium, mainly in phyllodes of the four Acacia species, and sometimes in branchlets and primary roots. These elements were most likely precipitated in forms of oxalate and sulfate in various tissues, including epidermis, mesophyll, parenchyma, sclerenchyma (fibre cells), pith, pith ray and cortex. In most cases, precipitation of calcium, magnesium, strontium and barium was biologically induced, and elements precipitated differed between soil types, plant species, and tissues within an individual plant; the precipitation was also related to tissue age. Formation of crystals containing these elements might play a role in regulating and detoxifying these elements in plants, and protecting the plants against herbivory.