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。     

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.     

Cell wall sheath surrounding calcium oxalate crystals in mulberry idioblasts

Summary. The distribution and ultrastructural features of idioblasts containing calcium oxalate crystals were studied in leaf tissues of mulberry, Morus alba L. In addition to the calcium carbonate crystals formed in epidermal idioblasts, large calcium oxalate crystals were deposited in cells adjacent to the veins and surrounded by a cell wall sheath which had immunoreactivity with an antibody recognizing a xyloglucan epitope. The wall sheath formation indicates exclusion of the mature crystal from the protoplast. Correspondence: Y. Sugimura, Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido, Matsugasaki, Sakyo, Kyoto 606-8585, Japan.     

堇菜叶片草酸钙晶体与水分维持的关系

随着全球气候变化加重,干旱强度和持续时间逐渐增加,严重影响植物生长和作物产量。喀斯特为典型的干旱和高钙生境,植物叶片富集大量的草酸钙晶体,而该晶体与植物耐旱性之间的关系并不清楚。该研究以喀斯特适生植物堇菜(Viola verecumda)为材料,土壤进行自然干旱,分析堇菜叶片的草酸钙晶体变化特征与水分之间的关系。结果表明:在土壤自然干旱条件下,堇菜主要通过细胞内束缚水的释放,维持细胞内水分平衡;而在干旱后期,叶片通过关闭气孔,将部分自由水转变为束缚水,防止水分流失。此外,草酸钙晶体的密度与束缚水含量具有极其显著的强正相关线性回归关系(r=0.825 3,P0.000 1),表明草酸钙晶体作为主要的束缚水物质。因此,堇菜植物在耐旱过程中可能协调草酸钙晶体和气孔的生理行为忍耐干旱胁迫。     

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.     

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。     

Production of Calcium Oxalate Crystals by the Basidiomycete Moniliophthora perniciosa, the Causal Agent of Witches’ Broom Disease of Cacao

Oxalic acid has been shown as a virulence factor for some phytopathogenic fungi, removing calcium from pectin and favoring plant cell wall degradation. Recently, it was published that calcium oxalate accumulates in infected cacao tissues during the progression of Witches’ Broom disease (WBD). In the present work we report that the hemibiotrophic basidiomycete Moniliophthora perniciosa, the causal agent of WBD, produces calcium oxalate crystals. These crystals were initially observed by polarized light microscopy of hyphae growing on a glass slide, apparently being secreted from the cells. The analysis was refined by Scanning electron microscopy and the compositon of the crystals was confirmed by energy-dispersive x-ray spectrometry. The production of oxalate by M. perniciosa was reinforced by the identification of a putative gene coding for oxaloacetate acetylhydrolase, which catalyzes the hydrolysis of oxaloacetate to oxalate and acetate. This gene was shown to be expressed in the biotrophic-like mycelia, which in planta occupy the intercellular middle-lamella space, a region filled with pectin. Taken together, our results suggest that oxalate production by M. perniciosa may play a role in the WBD pathogenesis mechanism.     

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.     

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

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

Calcium Oxalate Crystal Formation and Growth in Two Legume Species as Altered by Strontium

Young plants of Phaseolus and Canavalia were grown in nutrient solutions where calcium had been partially or totally replaced by either strontium or sodium. The deleterious effect of this replacement on growth and development was greater with strontium than with sodium. As the calcium content of the nutrient solution was decreased fewer calcium oxalate crystals were formed in leaf tissues. There were fewer crystals formed when calcium was replaced by strontium than by sodium. Changes in solubility characteristics of the crystals indicated that they had incorporated strontium.     

Cytoplasmic streaming of calcium oxalate crystals in Callipsygma wilsonis (Bryopsidales, Chlorophyta)

Light microscopic study of the giant‐celled, marine green alga Callipsygma wilsonis J. Agardh (Udoteaceae, Bryopsidales) revealed numerous birefringent crystalline inclusions in the terminal segments of the assimilatory axes. The inclusions were thin plates with a triangular shape in face view, a base up to 75 μm in length, and a height that was one‐seventh the length of the base. Crystals of various sizes commonly were stacked face‐to‐face with one or more edges coinciding, but removal of organic material by treatment in sodium hypochlorite resulted in disaggregation. The crystals were soluble in dilute hydrochloric acid without effervescence but were insoluble in acetic acid. These diagnostic chemical solubility tests and a positive reaction to the Yasue staining reaction indicated that the crystals were composed of calcium oxalate. Scanning electron microscopy showed that most crystals had smoothly curving edges, but some had truncate or beveled margins. Calcium oxalate crystals have been reported to occur in the large central vacuoles of several bryopsidalean species, but the crystals in C. wilsonis were present in the parietal cytoplasm, which was evident from the presence of crystals in streaming cytoplasm. Calcium oxalate crystals, amyloplasts, chloroplasts, and other cytoplasmic constituents moved along cytoskeletal cables at rates of approximately 2.8 μm s⁻¹. These findings add to a growing body of evidence that calcium oxalate crystals in diverse algae may be present in cellular compartments other than the central vacuole.     

ULTRASTRUCTURE OF SARCOPLASMIC RETICULUM PREPARATIONS

Fragmented sarcoplasmic reticulum (FSR) from rabbit muscle was examined by positive staining, negative staining, and freeze-etch electron microscopic techniques in the absence and presence of calcium transport conditions. The existence of 30–40 A particles covering the outer surface of FSR vesicles was confirmed by two different negative stains in unfixed, glutaraldehyde-fixed and osmium tetroxide-fixed material. Freeze-etch microscopy revealed a second type of particle, 80–90 A in diameter, on the fractured surfaces of FSR vesicles. Following calcium oxalate accumulation, negative and positive staining techniques provided evidence for large nodular deposits within FSR vesicles which probably correspond to calcium oxalate crystals and are responsible for increments in turbidity during calcium oxalate accumulation. The most probable configuration of FSR vesicles in solution is spherical. "Tadpole" or tubular configurations were not seen by freeze-etch microscopy, positive staining, or in prefixed negatively stained material.     

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。     

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 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     

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.     

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.     

Comparative studies on the invasion of cattle ticks (Rhipicephalus (Boophilus) microplus) and sheep blowflies (Lucilia cuprina) by Metarhizium anisopliae (Sorokin)

Microscopic investigations over time were carried out to study and compare the pathogenesis of invasion of ticks and blowflies by Metarhizium anisopliae. The scanning electron microscope and stereo light microscope were used to observe and record processes on the arthropods' surfaces and the compound light microscope was used to observe and record processes within the body cavities. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle, particularly the outer endocuticle. Although large numbers of conidia adhered to the surface of blowflies, no conidia were seen to germinate on external surfaces. A single germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggests that fungal invasion of the blowfly is primarily through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on tick cuticles, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16.     

THE ASSOCIATION OF DRUSE CRYSTALS WITH THE DEVELOPING STOMIUM OF CAPSICUM ANNUUM (SOLANACEAE) ANTHERS

Each of the two stomiums in the anther of Capsicum annuum (sweet pepper) consists of a single layer of cells immediately below the epidermis between two adjacent locules. Each stomium extends the entire length of the anther and splits open at pollen maturity. Many calcium oxalate druse crystals form within the vacuoles of the stomium cells in association with membrane complexes and paracrystalline bodies. These latter structures are reported here for the first time and each is considered to be a nucleation site for druse crystal formation. Prior to the appearance of membrane complexes and crystals within the vacuoles, plasmalemmasomes are visible next to the stomium cell walls and contain vesicles and fibrous material. We propose that these bodies carry wall materials, including calcium ions and possibly oxalate ions, into the vacuoles. Their presence coincides with crystal formation. Two other types of crystals occur in the connective tissue between stomiums and the single vascular strand. These crystals, along with those in the two stomiums, form at precise times during anther development. Contrary to the more numerous suggestions that crystals protect against predators or are metabolic waste products, we believe their formation aids in degradation and weakening of the cell walls between the locules and, thus, contributes to the release mechanism for the pollen.