Globoid Crystals Research Articles

Occurrence of Globoid Crystals in Cotyledonary Protein Bodies of Pisum sativum as Influenced by Experimentally Induced Changes in Mg, Ca and K Contents of Seeds

In many species globoid crystals in protein bodies of seeds are very common while in other species they are rarely observed. A review of literature suggested that the balance between divalent (Mg2+ and Ca2+) and monovalent (K+) cations may be important in determining whether or not globoid crystals will form. To test this hypothesis, experiments were carried out to add Mg and Ca, or Ca alone, to pods developing on K-deficient pea plants. While it was possible to cause a reduction in K concentration and increases in Mg and Ca concentrations, any changes to the normal mineral storage pattern in pea cotyledons were remarkably small. In some treatments, statistically significant increases in the ratio (Mg + Ca)/K were obtained and the sample with the greatest increase was examined in detail by electron microscopy and energy dispersive X-ray analysis. In this sample globoid crystals were common, in contrast to their normally rare occurrence in pea cotyledonary tissue.

Studies of Mineral Reserves in Pea (Pisum sativum) Cotyledons Using Low-Water-Content Procedures

Most of the phytin in pea (Pisum sativum) cotyledons is water soluble. In order to determine where K and P are located it was necessary to use anhydrous or low water content tissue preparation procedures to obtain samples suitable for energy dispersive X-ray analysis studies using a transmission electron microscope. While some protein bodies do contain electron-dense globoid crystals, most do not. Globoid crystals are more prevalent in the abaxial part of the cotyledon where the provascular network is located. When present, globoid crystals contain considerable Mg, and/or Ca along with P and K. Protein bodies that lack globoid crystals still contain considerable P and K with lesser amounts of elements such as S, Cl and Mg. This is consistent with these protein bodies containing K-phytate in the proteinaceous matrix. While there is a lot of K inside the protein bodies, K is widespread in pea cotyledon tissue and could be detected in starch grains, cell walls and the cytoplasmic matrix.

Protein body inclusions in seeds of Eucalyptus maculata and Eucalyptus erythrocorys

Polarizing light microscope studies of sections of embryo tissues revealed the presence of birefringent druse crystals in certain protein bodies of Eucalyptus erythrocorys and E. maculata. Light and electron microscope studies show that the location, size, and structure of the protein bodies that contain druse crystals differ in these two species. These species also differ in the number of druse-containing protein bodies occurring per cell and the size and structure of the druse crystals. Energy-dispersive X-ray analysis, acid solubility, and microincineration tests showed that these druse crystals are composed of calcium oxalate. In both species the druse crystals were retained by the seedling during germination and early seedling growth. Seeds of both Eucalyptus species also contain globoid crystals in most, nonprotodermal protein bodies.

Protein bodies in umbelliferous seeds. II. Elemental composition

The elemental composition of the protein bodies from several members of the Umbelliferae including carrot (Daucus carota L. cv. Imperator 408), wild carrot (Daucus carota L.), caraway (Carum carvi L.), anise (Pimpinella anisum L.), dill (Anethum graveolens L.), celery (Apium graveolens L. cv. Tall Utah), fennel (Foeniculum vulgare Mill.), parsnip (Pastinaca sativa L. cv. Hollow Crown), parsley (Petroselinum sativum L. cv. Moss Curled), and chervil (Anthriscus cerefolium L. cv. Curled) was determined using energy dispersive X-ray analysis. Globoid crystals in the endosperm usually contained P, K, and Mg or infrequently P, K, Mg, and Ca. In the embryos of carrot and caraway, P was always present with a combination of K, Mg, or Ca. Calcium was the only element detectable in the calcium-rich crystals. The proteinaceous matrix always contained S and K regardless of the inclusion present in the protein body.Quantitative determinations of P, Mg, K, and Ca are presented for all members studied except wild carrot, chervil, celery, and fennel. In all species analysed in this manner K or Ca were present in the highest amounts followed by P and Mg. Calcium-localization studies showed that most of the Ca is located in the endosperm and pericarp portions of the mericarp.

Protein bodies in umbelliferous seeds. I. Structure

The structure of the protein bodies from seeds of the family Umbelliferae has not been studied extensively since late in the 19th century. Using light and electron microscopy structural aspects of the protein bodies of carrot (Daucus carota L. cv. Imperator 408), wild carrot (Daucus carota L.), caraway (Carum carvi L.), anise (Pimpinella anisum L.), dill (Anethum graveolens L.), celery (Apium graveolens L. cv. Tall Utah), fennel (Foeniculum vulgare Mill), parsnip (Pastinaca sativa L. cv. Hollow Crown), parsley (Petroselinum sativum L. cv.Moss Curled), and chervil (Anthriscus cerefolium L. cv. Curled) were studied. Both endosperm and embryo protein bodies were investigated. Structurally, the protein bodies from all these genera were similar in that two types of protein bodies were found. One type consisted of a homogeneous, proteinaceous matrix and a number of variously sized, globoid crystal inclusions. The other type consisted of a homogeneous, proteinaceous matrix and either an individual or, more commonly, an aggregate of calcium-rich crystals arranged in a cluster usually termed a druse. Both types of protein bodies were never found in the same cell. Only globoid crystals were found in the embryo protein bodies. Protein bodies in the embryos were smaller, more numerous per cell, and often contained a flocculent, proteinaceous matrix.

Mineral Reserves in Castor Beans: The Dry Seed

Elemental composition and distribution of the mineral reserves in the endosperm and embryo tissues of Ricinus communis cultivars Hale and Zanzibarensis were investigated. Energy dispersive x-ray analysis was used to determine the elemental composition of the globoid crystals, while atomic absorption spectrometry allowed quantification of the elements, particularly Ca, in various seed regions. No major differences were found between the two cultivars with regard to the elemental distribution in globoid crystals. While the majority of globoid crystals contained P, K, and Mg, the occasional one also contained Ca. In extremely rare instances, Fe was detected in globoid crystals. Ca-containing globoid crystals were more common in provascular cell protein bodies in the stem and radicle. Polarized light microscopy, micro-incineration, and acid solubility tests demonstrated the presence of calcium oxalate crystals in the innermost testa which adheres to the endosperm and is often mistakenly identified as endosperm. Atomic absorption spectrometry revealed that most of the calcium present in castor bean seeds is localized in the testa. On a perseed-region basis, the much larger endosperm contains more Ca than does the embryo. However, on a unit-weight basis, the radicle-plus-stem regions contain considerably more Ca than does the cotyledon or endosperm, an observation that is consistent with the observed distribution pattern for Ca-containing globoid crystals.

Protein bodies in seeds

Membrane bound protein bodies (aleurone grains) are thought to be the main subcellular location of protein and mineral storage in seeds. In addition to structurally homogeneous proteinaceous matrix, protein bodies may contain protein crystalloids, electron–dense globoid crystals, electron–transparent soft globoids, and crystals of calcium oxalate. Protein crystalloids vary in shape, size and number. For example, cotyledon mesophyll cell protein bodies in the Cucurbitaceae generally contain protein crystalloids whereas those of Compositae and Cruciferae do not. Globoid crystals, which are rich in phytin, vary greatly in size and number per protein body. Some species have numerous small globoid crystals per protein body whereas others have one or two large globoid crystals per protein body. Phosphorus and various cations (K, Mg, Ca, Fe, Ba, Mn) located in globoid crystals can be studied with an energy dispersive X–ray (EDX) analysis system mounted on a transmission electron microscope. In some cases, cations such as Ca, Mn and Fe are specifically localized in globoid crystals of certain tissues or embryo regions. Further investigations may allow elemental composition of globoid crystals to be used in studies of systematics. Biref–ringent crystals, sometimes in the form of single large crystals but frequently in the form of druses, are present in protein bodies of some species. At least some endosperm protein bodies of all Umbelliferous species examined contain druse crystals. While seed protein bodies of relatively few species have been studied with electron microscopy, there are indications that protein bodies could be a useful character for studies in plant systematics.

Elemental composition of globoid crystals in protein bodies of wheat grain grown on soil treated with sewage sludge

Wheat grain grown on soil treated with a heavy application rate of sewage sludge exhibited higher iron, zinc, copper, and cadmium contents than did wheat grain grown on an unfertilized control treatment. The sludge supplied more than the maximum amounts of zinc, copper, cadmium, and lead recommended for addition to Ontario agricultural lands.Globoid crystals, which are inclusions in protein bodies, are the major mineral storage component of wheat grain. The elemental composition of globoid crystals in wheat grain grown with and without sludge treatment was determined using energy dispersive X-ray (EDX) analysis. Globoid crystals from sludge-treated and control grains exhibited similar elemental compositions with the following exceptions. When comparing sludge-treated grains versus the controls: more globoid crystals in the aleurone layer farthest from the peduncle contained calcium; more coleoptile mesophyll globoid crystals contained calcium, manganese, and (or) iron; some globoid crystals in the young foliage leaves contained manganese; and some globoid crystals in the stele of the radicle contained calcium. The globoid crystals exhibited no tendency to accumulate toxic metals supplied by the sludge indicating that the mineral storage system in wheat is remarkably specific despite varying soil conditions.

X-ray Analysis Studies of Elements Stored in Protein Body Globoid Crystals of Triticum Grains

Energy-dispersive x-ray analysis was used to investigate the elemental storage within protein bodies, specifically the globoid crystals, in grains of wheat. Areas of the grain investigated included various parts of the embryo, the aleurone layer plus starchy endosperm near the embryo and the aleurone layer plus starchy endosperm farthest from the embryo. Variations did occur grain-to-grain, cell-to-cell and, in certain regions, intracellularly. No protein bodies with electron-dense globoid crystals were found in the starchy endosperm. Generally globoid crystals contained P, K, and Mg in all areas investigated. Globoid crystals from the aleurone layer farthest from the embryo on occasion contained Ca, whereas aleurone globoid crystals near the embryo sometimes contained Fe. In most of the embryo regions examined, a few globoid crystals contained Ca along with P, K, and Mg. No specific pattern to the Ca distribution could be found. Welldefined elemental distribution occurred with Mn. Manganese was found only in globoid crystals located in the base and midregions of the stele in the radicle. Thus, in wheat there is some specific distribution of minerals dependent upon cell type and/or position in the grain.

Studies of metal uptake, especially iron, into protein bodies of Capsella and Lycopersicon seeds

Energy dispersive X-ray analysis was used to investigate metal uptake into the globoid crystals of Capsella and Lycopersicon seeds. Capsella seeds were taken from the curbside of a major roadway where metal contamination was highest, 5 m from the curb where less metal contamination occurs, and from an area well away from any roadways. Globoid crystals in Capsella protein bodies generally contained P, K, Ca, and Mg. No uptake of Pb or any other toxicant metals into the globoid crystals was found. Globoid crystals from the radicles and cotyledons of seeds produced by plants growing near the curb generally contained Fe whereas seeds of plants growing 5 m back from the curb contained traces of Fe in globoid crystals in certain tissues. Seeds of plants growing farthest from any roadway contained no Fe in their globoid crystals. Seeds of Lycopersicon were grown in metal-contaminated soil obtained near a smelter, a 1:1 mixture of this contaminated soil and regular potting soil, and a control. Globoid crystals from control seeds generally contained P, K, and Mg, while Ca, Mn, and (or) Fe were present occasionally. Control endosperm globoid crystals did not contain Fe. No pollutant metals were taken up into the globoid crystals. Iron which was present in the contaminated soil was found in the globoid crystals of the endosperm in those seeds obtained from plants grown in contaminated soil. Also, although Fe-containing globoid crystals were specifically located throughout the embryo in control seeds, there was no evidence that seeds of plants grown in contaminated soil contained more Fe in these areas.

Thin-section, freeze-fracture, and energy dispersive x-ray analysis studies of the protein bodies of tomato seeds

Protein bodies of dry seeds of tomato (Lycopersicon esculentum) from radicle, hypocotyl, cotyledon, and endosperm tissue were extensively studied using thin-sectioning, freeze-fracturing and energy dispersive x-ray (EDX) analysis. Protein bodies varied in size, were oval to circular in section, and generally consisted of a proteinaceous matrix, globoid crystal, and protein crystalloid components. Size, shape, and arrangements of globoid crystals and protein crystalloids varied even within the same cell. Globoid crystals were generally oval to circular in section. They were always surrounded by a proteinaceous matrix. In a given protein body the number present ranged from a few to numerous. A protein body generally contained only one protein crystalloid. In section, protein crystalloids were irregular or angular in shape. They were composed of substructural particles which formed lattice planes. EDX analysis of tomato seed globoid crystals revealed the presence of P, K, and Mg in all cases, a fact that is consistent with globoid crystals being phytin-rich. Rarely, small amounts of calcium were found along with P, K, and Mg in globoid crystals of each of the tissue regions considered. The distribution pattern of cells with Ca containing globoid crystals was random. Small amounts of Fe and Mn were also found in the globoid crystals of protein bodies from certain cell types. These two elements, unlike calcium, were specific in terms of their distribution. Globoid crystals from the protodermal cells often contained Mn and Fe. The globoid crystals from provascular tissue of radicle, hypocotyl, and cotyledon regions often contained Fe while globoid crystals in the first layer of large cells surrounding these provascular areas always contained Fe. Results from EDX analysis of the proteinaceous material from the protein bodies are presented and discussed as are variations in elemental content due to different fixations.

Calcium Distribution in Globoid Crystals of Cucurbita Cotyledon Protein Bodies

Energy-dispersive x-ray analysis was used to investigate the location of globoid crystals with relatively high Ca levels within cotyledons of Cucurbita maxima, Cucurbita mixta, and Cucurbita andreana. The small globoid crystals in both upper and lower epidermal cells commonly contained Ca. Ca was present in globoid crystals of all provascular regions with the exception of the very small provascular regions of C. maxima. In C. maxima and C. mixta cotyledons, some cases were observed where Ca was found in the globoid crystals of the first layer of mesophyll cells surrounding the provascular region, but in general Ca was absent from globoid crystals of palisade and spongy mesophyll cells. In C. andreana, globoid crystals of palisade and spongy mesophyll cells commonly contained at least some Ca. Cell position and cell type are factors affecting the Ca content of globoid crystals in protein bodies.

An energy dispersive x-ray analysis study of protein bodies in the seeds of Astragalus bisulcatus and Oxytropis Lambertii

Elemental composition of protein body regions in radicle, stem, and cotyledon areas of the leguminous plants Astragalus bisulcatus and Oxytropis Lambertii were determined using energy dispersive x-ray (EDX) analysis. Globoid crystals from all tissue regions of both species routinely contained P, K, Mg, and Ca. Possible relationships between seed size and the presence of calcium in globoid crystals of plants in the legume subfamily Papilionateae are discussed. Although the two species studied are selenium accumulators, no Se was ever detected in either globoid crystal or proteinaceous regions. The unusual mineral uptake properties of these plants seemingly does not influence the types of mineral reserves deposited in the protein bodies of the seed.

Composition of Globoid Crystals from Embryo Protein Bodies in Five Species of Cucurbita

Previous energy-dispersive x-ray analysis studies of globoid crystal composition in seed protein bodies gave an indication that there might be a correlation between seed size and the type of elements stored in globoid crystals. This possibility was tested by conducting energy-dispersive x-ray analysis studies of P, K, Mg, and Ca levels in globoid crystals of four embryo regions (radicle, stem, cotyledon center palisade mesophyll, cotyledon center spongy mesophyII) in each of five different Cucurbita species (C. mixta, C. moschata, C. foetidissima, C. pepo, and C. andreana). The species were chosen to provide a range of seed size and weight. Globoid crystals from all embryo regions in all five species contained P, K, and Mg. Some variations in the levels of these elements did occur but there was no consistent pattern with regard to area of the seed or with regard to seed size. Calcium distribution showed significant variations. In species with large seeds (C. mixta, C. moschata) Ca was mainly found in globoid crystals in the radicle. Globoid crystals in species with small seeds (C. foetidissima, C. pepo, C. andreana) contained Ca in all embryo regions tested. The results of this study support the concept that Ca distribution in globoid crystals can be correlated with seed weight.

An energy dispersive x-ray analysis study of elemental loss from globoid crystals in protein bodies as a result of osmium tetroxide fixation

This study was undertaken to discover what elemental losses, if any, were occurring from globoid crystals in seed protein bodies during glutaraldehyde – osmium tetroxide fixation. Unfixed cotyledon and radicle tissue of Cucurbita maxima seed, or tissue after glutaral–dehyde–OsO4 treatment, was quick frozen in liquid N2, ground with a cold mortar and pestle, and low-temperature freeze-dried. Globoid crystals in the freeze-dried powder were subjected to energy dispersive x-ray (EDX) analysis. OsO4 fixation resulted in a major loss of P, Mg, and K from cotyledon globoid crystals and a major loss of P, Mg, K, and Ca from radicle globoid crystals. Despite the loss of elements, the OsO4-fixed globoid crystals were still electron dense. When globoid crystals from glutaraldehyde-fixed, dehydrated, and embedded cotyledon tissue were compared with globoid crystals from glutaraldehyde–OsO4-fixed, dehydrated, and embedded tissue, some extraction was found. The degree of extraction varied from complete loss of P, K, and Mg to loss of K only.Effects of glutaraldehyde–OsO4 fixation upon elemental composition of globoid crystals in several other species was also determined. Brazil nut (Bertholletia excelsa) radicle tissue or cotyledon tissue from walnut (Juglans regia), hazelnut (Corylus avellana), sunflower (Helianthus annuus), golden everlasting daisy (Helichrysum bracteatum), cashew (Anacardium occidentale), pistachio (Pistacia vera), and the Western Australian red-capped gum (Eucalyptus erythrocorys) were fixed either in glutaraldehyde or in glutaraldehyde–OsO4. In these species, EDX analysis of globoid crystal sections showed that OsO4 fixation results in major loss of Mg, K, and Ca. Generally, phosphorus levels were reduced from control values as well. When carrying out EDX analysis studies of globoid crystals, we recommend (1) avoiding any use of OsO4, (2) keeping all fixation, washing, and dehydration times as short as possible, and (3) checking all observations with freeze-dried powders.

Location of reserves of mineral elements in seed protein bodies: macadamia nut, walnut, and hazel nut

This paper presents the results of thin-section, freeze-fracture, energy dispersive x-ray (EDX) analysis, and chemical analysis studies of cotyledon tissue from dry seeds of macadamia nut (Macadamia integrifolia), walnut (Juglans regia), and hazel nut (Corylus avellana). Studies concentrated on mineral elements in the storage protein bodies. Protein bodies of macadamia nut contained no protein crystalloids but did contain some globoid crystals. Aqueous fixation of macadamia tissue appeared to result in a rapid solubilization of proteinaceous matrix material. EDX analysis of macadamia nut globoid crystals revealed the presence of P, K, and Mg in most cases but the occasional crystal was very high in calcium. Walnut protein bodies often contained many large protein crystalloids. Globoid crystals were present between the protein crystalloids and in the proteinaceous matrix. EDX analysis indicated the presence of P, K, and Mg as the main elements in walnut globoid crystals. Hazel nut protein bodies contained one or more small protein crystalloids, globoid crystals of various sizes, and proteinaceous matrix. Some protein bodies contained druse crystals. EDX analysis showed that the druse crystals were rich in calcium and thus are presumably calcium oxalate. EDX analysis of globoid crystals revealed the presence of elements consistent with the phytin-rich nature of globoid crystals. Variations in globoid crystal composition are discussed.

Thin sectioning, freeze fracturing, energy dispersive x-ray analysis, and chemical analysis in the study of inclusions in seed protein bodies: almond, Brazil nut, and quandong

Protein bodies from almond (Prunus dulcis), Brazil nut (Bertholletia excelsa), and quandong (Santalum acuminatum) have been studied in thin sections of fixed and embedded tissue, in freeze-fracture replicas of unfixed tissue, by chemical analysis of tissue for P, K, Mg, and Ca, and by energy dispersive x-ray (EDX) analysis of both sections of glutaraldehyde-fixed tissue and freeze-dried tissue powders. The protein bodies in all three species contained globoid crystals, protein crystalloids, and proteinaceous matrix regions. Results of EDX analyses were consistent with globoid crystals being rich in phytin. Variation in both the structure and the elemental composition of globoids was common. In almond some globoids were lobed rather than spherical, and large globoid crystals often contained considerable calcium whereas small globoid crystals contained little if any calcium. The globoid crystals of Brazil nut often contained barium in addition to P, K, Ca, and Mg. Protein crystalloids of Brazil nut were compound crystals. Protein bodies of quandong seed, which is largely endosperm rather than embryo, were unexceptional.

Calcium oxalate druse crystals and other inclusions in seed protein bodies: Eucalyptus and jojoba

Druse crystals of calcium oxalate are known to occur in some protein bodies of some plant seeds. This paper reports observations on crystals, and other contents, of protein bodies of Eucalyptus erythrocorys and Simmondsia chinensis (jojoba). Results are presented from thin section studies of glutaraldehyde–OsO4 fixed, dehydrated, and embedded tissue; freeze-fracture studies; energy dispersive x-ray (EDX) analysis studies of freeze-dried tissue powders (Eucalyptus only); and chemical analysis studies of P, Mg, K, and Ca content (Eucalyptus only). Many Eucalyptus protein bodies contained large druse crystals rich in Ca but devoid of P, K, and Mg, and an occasional protein body from jojoba contained some dispersed crystals. Eucalyptus seeds were exceptionally high in Ca content. EDX analysis results provide evidence favouring the phytin-rich nature of globoid crystals in the two species. Structural variation in the globoid crystals was great, especially in jojoba. Eucalyptus, whose protein bodies contained very large globoid crystals rich in P, Mg, and K, was found to have higher levels of these elements than most species investigated to date.

Inclusions in seed protein bodies in members of the Compositae and Anacardiaceae: comparison with other dicotyledonous families

Thin sections, freeze-fracture replicas, energy dispersive x-ray (EDX) analyses, and chemical analyses of cotyledon tissue from seeds of sunflower (Helianthus annuus), golden everlasting daisy (Helichrysum bracteatum), cashew (Anacardium occidentale), and pistachio nut (Pistacia vera) showed that in all four species the protein bodies consisted of a proteinaceous matrix surrounding numerous globoid crystals. Some variation in globoid crystal size did occur, but they were generally small in relation to the size of the protein body. No protein crystalloids were found in the cotyledon protein bodies of these four species. EDX analysis results were consistent with the globoid crystals being phytin rich. Results of this study, combined with an evaluation of protein body structure in other dicot embryos that have been studied, support the theory that protein body structure is related to taxonomic grouping.

Energy-dispersive x-Ray Analysis of Phosphorus, Potassium, Magnesium, and Calcium in Globoid Crystals in Protein Bodies from Different Regions of Cucurbita maxima Embryos

The seeds of Cucurbita maxima contain protein bodies with electrondense globoid crystals. Because of their density globoid crystals are ideal material for energy-dispersive x-ray (EDX) analysis studies of elemental composition. Fixation trials were carried out to test globoid crystal extraction during glutaraldehyde fixation, water washing, and ethanol dehydration. Glutaraldehyde fixation without subsequent washing or dehydration alone produced no significant changes in elemental composition of cotyledon globoid crystals. If glutaraldehyde fixation was followed by water washes or ethanol dehydration there was some loss of the major globoid crystal elements but the relative percentages of the elements P, K, Ca, and Mg remained relatively unchanged. In this paper results of a study of the P, K, Mg, and Ca content of globoid crystals in different tissues of squash embryos are presented. The globoid crystals in the radicle were found to be the least dense in the embryo. Globoid crystals from all embryo regions contained P, K, and Mg. In the various embryo regions P and Mg maintained relatively constant proportions of the globoid crystal composition while K and Ca varied. Of particular significance is the distribution of Ca which is generally an immobile element. Calcium was found in highest amounts in the globoid crystals of the radicle and stem regions while globoid crystals in much of the cotyledon contained little, if any, Ca. The Ca storage thus seems to be spatially arranged in a manner that would aid early growth of the root-shoot axis.