Cytological changes during cell wall sac formation in mulberry idioblasts

Summary. When calcium carbonate crystals are formed in mulberry (Morus abla) idioblasts, they are deposited in newly formed cell wall sacs. The initial cytological events leading to cell wall sac formation were observed in the distal end of young idioblasts and tentatively categorized into four stages. The first indication of formation was the separation of the innermost cell wall layer from the cell wall, which is followed by the deposition of egg-shaped polysaccharide on the inner cell wall surface. The size of the deposit area increased, while the thickness of the cell wall significantly decreased during the next stage. Finally, the condensed cellulosic lamella was invaginated into the deposition area, resulting in the formation of an elongated cell wall sac. The internal cell wall sac was composed of numerous fibers with different morphologies. Application of gelatin-methenamine-silver staining allowed us to observe the spatial distribution of cellulosic polysaccharides as electron-dense images. Correspondence and reprints: Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan.     

Calcium carbonate deposition in a cell wall sac formed in mulberry idioblasts

Summary. Although calcium carbonate is known to be a common biomineral in plants, very little attention has been given to the biological control of calcium carbonate deposition. In mulberry leaves, a subcellular structure is involved in mineral deposition and is described here by a variety of cytological techniques. Calcium carbonate was deposited in large, rounded idioblast cells located in the upper epidermal layer of mulberry leaves. Next to the outmost region (“cap”) of young idioblasts, we found that the inner cell wall layer expanded to form a peculiar outgrowth, named cell wall sac in this report. This sac grew and eventually occupied the entire apoplastic space of the idioblast. Inside the mature cell wall sac, various cellulosic membranes developed and became the major site of Ca carbonate deposition. Concentrated Ca²⁺ was pooled in the peripheral zone, where small Ca carbonate globules were present in large numbers. Large globules were tightly packed among multiple membranes in the central zone, especially in compartments formed by cellulosic membranes and in their neighboring membranes. The maximum Ca sink capacity of a single cell wall sac was quantified using enzymatically isolated idioblasts as approximately 48 ng. The newly formed outgrowth in idioblasts is not a pure calcareous body but a complex cell wall structure filled with substantial amounts of Ca carbonate crystals. Correspondence and reprints: Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido, Matsugasaki, Sakyo, Kyoto 606-8585, Japan.     

Ultrastructure of the suberized styloid crystal cells in Agave leaves

Summary Styloid calcium oxalate crystal idioblasts of Agave americana L. are suberized. Where the crystals do not touch the cell wall directly they are enclosed in a suberinic sheath which is connected with the suberinic wall layer. No polysaccharides are laid down as a tertiary wall layer, nor could any polysaccharides be found in the crystal sheath. These results contradict those of Arnott (1973) but agree fully with those of Rothert and Zalenski (1899).     

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。     

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     

A calcium oxalate-secreting tissue in branchlets of the Casuarinaceae

Summary Deciduous branchlets of casuarina trees have an unusual calcium oxalate-secreting system in which the epidermal tissue deposits calcium oxalate crystals in cell walls of the branchlet surface. These prismatic crystals were identified by light and electron microscopy, histochemistry, and elemental X-ray analysis. This calcium oxalate-secreting tissue was found in all species of casuarinas examined, including three of the four genera of the Casuarinaceae:Allocasuarina sp.,Casuarina sp., andGymnostoma papuanum. Because crystals were present throughout the epidermis soon after it formed, the mechanism for their induction was likely to be different than that for calcium oxalate crystal idioblasts. Secreting cells had a complex endoplasmic reticulum that may be involved in the secretory process.Abbreviations EDS energy-dispersive X-ray spectroscopy - HPF/FS high pressure-frozen/freeze-substituted - SEM scanning electron microscopy - TEM transmission electron microscopy Dedicated to the memory of Professor John G. Torrey     

Calcium oxalate crystals and crystal cells in the leaves ofRhynchosia caribaea (Leguminosae: Papilionoideae)

Summary The development and distribution of calcium oxalate crystals, stomates and hairs were studied in the first trifoliolate leaf ofRhynchosia caribaea (Leguminosae: Papilionoideae, Phaseoleae). Using light and transmission electron microscopy, the crystals were shown to occur in both bundle sheath and mesophyll cells. Crystal distribution and shapes are characteristic forRhynchosia. Crystals develop late in leaf development in contrast to the stomates and hairs. As these latter two structures decrease in number per unit area with leaf age, crystal number increases.     

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

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

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。     

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.     

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。     

Incorporation of strontium into plant calcium oxalate crystals

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

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

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

ASSOCIATION OF FOUR DIFFERENT CALCIUM CRYSTALS IN THE ANTHER CONNECTIVE TISSUE AND HYPODERMAL STOMIUM OF CAPSICUM ANNUUM (SOLANACEAE) DURING MICROSPOROGENESIS

The anther connective tissue and hypodermal stomium between adjacent locules in the anthers of Capsicum annuum L. (Solanaceae) are the sites of formation of calcium salt crystals with four different habits. The spatial and temporal associations of these crystals and the idioblastic cells in which they form indicate that crystal sand occurs earliest in anther development near the single vascular strand, followed by spherulites and prismatic crystals farther out in the connective tissue, and finally druses occur in the hypodermal stomium. Both the druses and the crystal sand crystals are encased in crystal chambers and are associated with distinct membranes, whereas the spherulites and prismatic crystals are not bounded by any apparent membranes but they are surrounded by dense material that is rich in calcium and stains positively for polysaccharides and proteins. Quite often spherulites and prismatic crystals are observed within a single cell in contact with each other. X-ray diffraction of crystal preparations containing all four crystal habits and X-ray elemental analyses of single crystals, as well as visual observations and acid treatments, suggest that all four crystal habits consist of calcium oxalate. The hypodermal stomium and adjacent connective tissue degenerate at the pollen stage causing adjacent locules to fuse. Shortly afterward, each stomium epidermis splits open along the length of the anther releasing the pollen. It is suggested that the crystal idioblasts are involved in this process, possibly by a temporally orchestrated sequestration of calcium from both the cell cytoplasm and cell wall.     

Calcium and oxalate content of the leaves of Phaseolus vulgaris at different calcium supply in relation to calcium oxalate crystal formation

Soluble and insoluble oxalate and insoluble calcium were measured in the leaves of Phaseolus vulgaris. The plants were grown in nutrient solutions with two different concentrations of calcium. Two developmental stages of the leaves were studied. Although the content of insoluble calcium differs widely according to leaf age and growth conditions, the percentage bound in crystals is nearly the same in all cases. In the growing leaves, concentrations of total oxalate are independent of calcium supply, thus, showing that the known rise in numbers of crystals, and of cells containing them, is not induced via oxalate biosynthesis. Fully expanded leaves contain more oxalate when grown in a nutrient solution with higher calcium concentration. Amounts of oxalate in percent of dry weight are similar to those given in the literature for other legume leaves.     

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.     

Ultrastructural and immunochemical features of the cell wall sac formed in mulberry (Morus alba) idioblasts

A peculiar inward growth, named a “cell wall sac”, formed in mulberry (Morus alba) idioblasts, is a subcellular site for production of calcium carbonate crystals. On the basis of ultrastructural observations, a fully expanded cell wall sac could be divided into two parts—an amorphous complex consisting of multi-layered compartments with multiple fibers originating from the innermost cell wall layer, and a peripheral plain matrix with fiber aggregates. Immunofluorescent localization showed that low and highly esterified pectin epitopes were detected at the early stages of development of the cell wall sac, followed by complete disappearance from the both parts of fully enlarged mature sac. In contrast, the xyloglucan epitope remained in the compartment complex; this was supported by the observation that the xyloglucan epitope labeled with immuno-gold particles is found on fibers in the complex part.     

Structure of Crystal Cells and Influences of Leaf Development on Crystal Cell Development and Vice Versa in Phaseolus vulgaris (Leguminosae)

The structure of cells with calcium oxalate crystals and their nelghbouring cells has been studied by light and transmission electron microscopy at different stages of bean leaf development. Plants were grown with varying calcium supply to identify a possible influence of calcium nutrition on cell structure. Crystals are formed inside the vacuole of already highly vacuolated cells of bundle sheath extensions. The membrane around the crystal vacuole is continuous with the plasmalemma. The crystal vacuole contains membraneous structures. In the fully expanded leaf the crystal becomes ensheathed by wall material. Chloroplasts of bundle sheath extension cells, with or without crystals, are smaller, with fewer membranes, and with much narrower stroma regions than those of the palisade parenchyma. There is a stage in the young leaf when only the bundle sheath extension cells without crystals have starch grains in their chloroplasts. As their number is lower in plants grown with high calcium supply this means that, in this case, less cells are competent for photosynthesis.     

Calcium Oxalate Raphide Crystals and Crystalliferous Idioblasts in the Carpels of Ornithogalum caudatum

Crystalliferous idioblasts commonly are found in groups of twoor three cells in the peripheral region of the carpels Crystals,composed of calcium oxalate, usually are m well-organized bundleswhich develop within a matrix of protein and carbohydrate inthe vacuole of each idioblast The matrix occurs around and betweenindividual crystal chambers and contains spheres and tubules5.4 nm in diameter The matrix changes in character and locationwith age Crystals form within their own individual chambers,each comprised of a series of lamellae The number of lamellaeis variable The innermost lamella is different from the othersin that it is apparently continuous The other lamellae are platelikeand superficially resemble successive periderms. The lamellaemay begin and/or terminate abruptly or they may anastamose Eachlamella is composed of chains of spheres about 6 1 nm in diameterand is separated from adjacent lamellae by tubules 5.4 nm indiameter Both the crystals and slime body are absorbed duringlater stages of carpel maturation. Ornithogalum caudatum Ait carpel, calcium oxalate, idioblasts, ultrastructure     

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

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