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

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

植物晶体的形态结构、生物功能及形成机制研究进展

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

Diversity and distribution of idioblasts producing calcium oxalate crystals in Dieffenbachia seguine (Araceae)

Although cells that synthesize crystals are known throughout the plant kingdom, their functional significance is still unknown. Mechanical support, mineral balance, waste sequestration, and protection against herbivores have all been proposed as crystal functions. To seek clues to their role(s), I systematically examined all organs except fruit of Dieffenbachia seguine (Araceae) for crystals. Crystals were found in nearly every organ. Raphides (long, slim, pointed crystals) were most common, but druses (crystal aggregates) and prisms were also found. Raphides varied in size by a factor of 10 and also in organization from tightly bundled to loosely organized. Biforines, a type of cell capable of expelling raphides, or biforine-like cells, were found in nearly all organs, but especially in leaves, spathes, and anthers. Different organs had different crystal complements, and characteristic crystals were found at specific locations, such as among pollen, along the undersides of leaf veins, and at root branch points. All crystals appeared to be composed of calcium oxalate, based on acid solubility. Possible roles of the crystals are discussed in light of these findings.     

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。     

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.     

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.     

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

Background and Aims

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

Methods

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

Key Results

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

Conclusions

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

Engineering calcium oxalate crystal formation in Arabidopsis

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

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

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

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.     

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.     

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     

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.     

Twinned Raphides of Calcium Oxalate in Grape (Vitis): Implications for Crystal Stability and Function

Among the higher plants that accumulate crystalline calcium oxalate, many taxa characteristically produce raphides, or needle-shaped crystals. Substantial evidence has accumulated indicating that raphides function in plant defenses against herbivory and that their acicular shape is a critical component in proposed mechanisms for these defenses. Previous observations have shown that raphides in members of the Vitaceae are twinned crystals. In this study, the fine structure of raphides in Vitis was examined in order to determine the nature of twinning. Rotation of isolated raphides under cross-polarized light revealed extinction patterns that indicated that raphides are twinned along their length. In cross sections of raphides, the twin plane extends across the raphides, parallel to their surfaces. The dissolution patterns observed in etched crystals indicate that the type of twinning is rotational. Parallels in other biomineralization systems indicate that the rotational nature of the twinning may increase mechanical strength. In addition, because twinning may affect crystal growth or enhance stability and persistence of crystals, it could be an important factor in allowing plant cells to produce the raphide morphology.     

Isolation of Medicago truncatula mutants defective in calcium oxalate crystal formation

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

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.     

Plant calcium oxalate crystal formation, function, and its impact on human health

Crystals of calcium oxalate have been observed among members from most taxonomic groups of photosynthetic organisms ranging from the smallest algae to the largest trees.The biological roles for calcium...     

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 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.     

OCCURRENCE,TYPE, AND LOCATION OF CALCIUM OXALATE CRYSTALS IN LEAVES AND STEMS OF 16 SPECIES OF POISONOUS PLANTS

Crystals in 16 species of poisonous plants growing naturally in Saudi Arabia were studied with light microscopy. Three types of crystals were observed: druses, prismatics, and crystal sand. Raphides and styloids were not observed in any of the species studied. Druses occur more frequently in the leaf midrib and in the cortex and pith of the stem. In contrast, crystal sand and prismatic crystals are rare and occur in the leaf, intercostal lamina, and in the vascular tissues. The preliminary results show the absence of the three types of calcium oxalate crystals in the stem and leaf of seven species: Ammi majus L., Anagallis arvensis L., Calotropis procera Ait., Citrullus colocynthis (L.) Schard, Euphorbia peplis L., Hyoscyamus muticus L., and Solarium nigrum L., and the presence of druses, prismatic crystals, and crystal sand either in the leaf and stem or in the leaves or stems of nine species: Anabasis articulata (Forssk.) Moq. in DC., Chenopodium album L., Convolvulus arvensis L., Datura stramonium L., Nerium oleander L., Ricinus communis L., Rumex nervosus Vahl., Pergularia tomentosa L., and Withania somnifera (L.) Dun. in DC. These observations indicate that there is no apparent relationship between the distribution of calcium oxalate crystals and the toxic organs of the plants, and supports the view that the presence of calcium oxalate crystals may not be related to plant toxicity.